DNA vaccination for treatment of autoimmune disease

ABSTRACT

A pro-inflammatory T cell response is specifically suppressed by the injection into a recipient of DNA encoding an autoantigen associated with autoimmune disease. The recipient may be further treating by co-vaccination with a DNA encoding a Th2 cytokine, particularly encoding IL4. In response to the vaccination, the proliferation of autoantigen-reactive T cells and the secretion of Th1 cytokines, including IL-2, IFN-γ and IL-15, are reduced.

CROSS-REFERENCES

[0001] This application claims priority to earlier filed InternationalPatent Application Ser. No. PCT/US00/06233, filed Mar. 10, 2000 whichapplication claims priority to U.S. patent application Ser. No.09/267,590 filed Mar. 12, 1999, both of which applications areincorporated herein by reference in their entirety.

INTRODUCTION

[0002] The complexity of the immune system has been a daunting barrierto an understanding of immune system dysfunction. In recent years, thetechniques of molecular biology have provided insight into themechanisms and components that underlie immunity. To a large extent, thestory of immunity is the story of lymphocytes. Lymphocytes possess anextremely complex and subtle system for interacting with each other,with antigen-presenting cells, and with foreign antigens and cells.

[0003] Modulation of the immune response varies with the specificfactors produced, and the receptors present on the responding cell. Thepathways for down-regulating responses are as important as thoserequired for activation. T cell tolerance is one well-known mechanismfor preventing an immune response to a particular antigen. Othermechanisms, such as secretion of suppressive cytokines, are also known.

[0004] A common feature in a number of diseases and inflammatoryconditions is the involvement of pro-inflammatory CD4⁺ T cells. These Tcells are responsible for the release of inflammatory, Th1 typecytokines. Cytokines characterized as Th1 type include interleukin 2(IL-2), γ-interferon, TNFα and IL-12. Such pro-inflammatory cytokinesact to stimulate the immune response, in many cases resulting in thedestruction of autologous tissue. Cytokines associated with suppressionof T cell response are the Th2 type, and include IL-10, IL-4 and TGF-β.It has been found that Th1 and Th2 type T cells may use the identicalantigen receptor in response to an immunogen; in the former producing astimulatory response and in the latter a suppressive response.

[0005] Cytokines play a critical role in the development and recoveryfrom autoimmune diseases. Th1 cytokines such as interleukin 12 (IL-12)and interferon gamma (IFNγ) have been found in the central nervoussystem (CNS) of multiple sclerosis (MS) patients as well as in animalswith EAE (Issazadeh et al. (1995). J Neuroimmunol 61:205-12). Th2cytokines such as IL-4, IL-5 and IL-10 have been found to be elevatedeither during remission of MS or EAE (Waisman et al. (1997)Immunointervention in autoimmunity by Th1/Th2 regulation, L. Adorini,ed. (Austin, Tex.: R.G. Landes Co.), pp. 129-50). Previous studies haveshown that systemic administration of IL4 as well as local CNSadministration of IFNγ can reduce the severity of EAE (Racke et al.(1994) J Exp Med 180:1961-6; Voorthuis et al. (1990) Clin Exp Immunol81:183-8). Furthermore, the addition of IL-4 to naive T cells can resultin the development of Th2 type cells, whereas the addition of IL-12 canresult in the development of Th1 type cells (Macatonia et al. (1993) IntImmunol 5:1119-28).

[0006] DNA vaccination is effective in protecting experimental animalsagainst infectious pathogens and cancer, and recently has been used toprevent autoimmune disease (Waisman et al. (1996) Nat Med 2, 899-905).Experimental autoimmune encephalomyelitis (EAE), a prototypic animalmodel of T cell autoimmunity, reflects many of the clinical andpathologic features of the human disease, multiple sclerosis.

[0007] In order to modify immune responses to DNA vaccines, DNAco-vaccination has been performed with cytokine genes, along with thegenes for certain pathogens. Examples include DNA immunization withhepatitis B virus antigens and IL-2 DNA which enhanced TH1 responses,HIV antigens with IL-12 DNA which enhanced cytotoxic T cell activity,and influenza antigens with IL-6 DNA which enhanced anti-viral activity(see, for example, Chow et al. (1998) J Immunol 160(3):1320-9).

[0008] Vaccination of mice with naked DNA that encodes the predominant Tcell receptor (TCR) β chain that is rearranged in myelin basic protein(MBP) reactive T cells, has been shown to protect mice from EAE. Suchimmunization induced a pattern of Th2 cytokine production by myelinreactive T cells, creating a suppressive environment blockingautoimmunity: T cells reacting to the myelin autoantigen deviated froman aggressive T helper 1 (Th1) type to a suppressive Th2 type.

[0009] Further development of treatment that specifically inhibits Tcell activation would be of great medical benefit.

[0010] Relevant Literature

[0011] Waisman et al. (1996) Nat. Med. 2:899-905 and) Offner et al.(1998) J. Immunol. 161:2178-2186 describe the use of DNA vaccination toprevent experimental autoimmune encehalomyelitis (EAE). The injection ofDNA to promote vaccination against microbes and tumors is discussed inCohen et al. (1997) Hosp. Pract. 32:169-171; Syrengelas, et al. (1996)Nat. Med. 2:1038-1041; Ulmer et al. (1996) Curr Opin Immunol.8:531-536.; Pardoll et al. (1995) Immunity 3:165-169; Davis et al.(1993) Hum. Mol. Genet. 2:1847-1851; Ulmer et al. (1993) Science259:1745-1749; and Tang et al. (1992) Nature 356:152-154. Geneticimmunization has demonstrated induction of both a specific humoral butalso a more broadly reacting cellular immune response in animal modelsof cancer, mycoplasma, TB, malaria, and many virus infections, includinginfluenza and HIV. See, for example, Mor et al. (1995) J Immunol155:2039-46; Xu and Liew (1995) Immunology 84:173-6; and Davis et al.(1994) Vaccine 12:1503-9.

[0012] Susceptibility to multiple sclerosis (MS) has been associatedwith certain MHC Class II genes, Oksenberg and Steinman (1990) CurrentOpinion in Immunology 2:619-621. At the cellular level, oligoclonalityof T-cells has been described in the cerebrospinal fluid (CSF) of MSpatients, Lee et al., Ann. Neurol. 29:33-40 (1991).

[0013] CNS antigens, including myelin proteins, studied in the contextof MS are discussed in de Rosbo et al., J. Autoimmunity 11:287-299(1998). Enhancers of the immune response to DNA vaccines includeunmethylated CpG dinucleotides, Krieg et al. (1998) Trends Microbiol.6:23-27, and fused pathogen-derived sequences, King et al. (1998) Nat.Med. 4:1281-1286.

SUMMARY OF THE INVENTION

[0014] Methods are provided for the suppression of pro-inflammatory Tcell responses in autoimmune disease. A mammalian host is vaccinatedwith a DNA expression vector encoding an autoantigen fragment. Inresponse to the vaccination, pathogenic T cell proliferation isinhibited and production of Th1 cytokines, including IL-2, IL-10, IFN-γand IL-15 is reduced. In one embodiment of the invention, a nucleic acidencoding a Th2 cytokine is co-administered with the autoantigen codingsequence. The use of IL-4 coding sequences is of particular interest.Suppressive vaccination diminishes T cell pro-inflammatory responses ina specific, targeted manner. Conditions that benefit from this treatmentinclude autoimmune diseases, tissue transplantation and other diseasesassociated with inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1. Anti-SCH IgG (A) and anti-PLP139-151 (B) antibody titersin SJL/J mice after DNA immunization with the PLP minigene.

[0016]FIG. 2. Lymph node cell proliferative responses to PLP139-151(squares) and the control peptide PLP178-191 (triangles) for animalsinjected with DNA coding for PLP139-151 (A) or control vector, pTARGET(B).

[0017]FIG. 3. (A) Levels of γ-interferon (striped bars) or IL-2 (dottedbars) in animals vaccinated with plasmid DNA coding for PLP139-151 orvector alone (pTARGET). (B) Cytokine mRNA detection and analysis by 5%polyacrylamide gel electrophoresis.

[0018]FIG. 4. Surface expression of B7.1, B7.2, and I-A^(s) of spleencells after incubation with DNA. Numbers in quadrants refer to thepercentage of cells in the monocyte gate (A) or the lymphocyte gate (B)as defined by forward and side scatter.

[0019]FIG. 5. Incidence of diabetes in DNA vaccinated NOD mice. FemaleNOD mice were injected with either empty plasmid DNA (▴), plasmidencoding insulin B (9-23) (▪), or plasmid encoding insulin A (7-21) (♦);one group was left untreated ().

[0020]FIG. 6. Quantitative PCR measurement of cytokine expression bypancreatic lymph node cells from vaccinated NOD mice cultured with 10μg/ml insulin B (9-23) peptide. PcDNA control vaccinated levels (solidbars) were used as a standard against which the insB-PcDNA vaccinatedvalues (hatched bars) were compared.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0021] The subject methods provide a means for therapeutic treatment andinvestigation of inflammation, through the suppression of pathogenicantigen-specific T-cell responses. A DNA expression cassette is injectedinto host tissue, for example muscle or skin. The vector comprises a DNAsequence encoding at least a portion of an autoantigen. The vaccinationmay also include DNA sequences encoding a Th2 cytokine, e.g. IL-4. Inresponse to this vaccination, a suppressive response is evoked.Antigen-specific T cell proliferation is inhibited and Th1 cytokineproduction is reduced.

[0022] Without limiting the scope of the invention, it is believed thatthe methods described herein are a novel method of protective immunity,which combines the effects of DNA vaccination and local gene delivery.After DNA vaccination with a autoantigen epitope alone, T cells areanergic. This may be in part due to the biological effects of DNA motifslike unmethylated CpG dinucleotides in particular base contexts (CpG-Smotifs) (Krieg et al. (1998) Trends in Microbiol. 6:23-27). The additionof IL4 as a DNA co-vaccine rescues the anergy imposed by the autoantigenDNA vaccine, and drives the response to a Th2 phenotype. STAT6 isactivated in draining lymph node cells by the IL4 DNA vaccine. It isbelieved that IL4 is produced from the DNA vaccine administered and thatit interacts with IL4 receptor on lymph node cells, which in turn causesthe activation of STAT6 downstream of the receptor. Immunization againstthe antigens that trigger those autoimmune diseases caused by Th1autoreactive cells, diseases such as multiple sclerosis, juvenilediabetes and rheumatoid arthritis, would be conditions whereco-vaccination with DNA encoding IL-4 might prove beneficial

[0023] Autoantigens, as used herein, are endogenous proteins orfragments thereof that elicit a pathogenic immune response. Ofparticular interest are autoantigens that induce a T cell mediatedinflammatory pathogenic response. Suppressive vaccination with therelevant target autoantigen finds use in the treatment of autoimmunediseases characterized by the involvement of pro-inflammatory T cells,such as multiple sclerosis, experimental autoimmune encephalitis,rheumatoid arthritis and insulin dependent diabetes mellitus. Animalmodels, particularly small mammals, e.g. murine, lagomorpha, etc. are ofinterest for experimental investigations.

[0024] The subject methods of suppressive immunization are used forprophylactic or therapeutic purposes. Use used herein, the term“treating” is used to refer to both prevention of disease, and treatmentof pre-existing conditions. The prevention of autoimmune diseaseinvolving the vaccine autoantigen (VA), is accomplished byadministration of the vaccine prior to development of overt disease. Thetreatment of ongoing disease, where the suppressive vaccinationstabilizes or improves the clinical symptoms of the patient, is ofparticular interest. Such treatment is desirably performed prior tocomplete loss of function in the affected tissues.

[0025] Autoantigens known to be associated with disease include myelinproteins with demyelinating diseases, e.g. multiple sclerosis andexperimental autoimmune myelitis; collagens and rheumatoid arthritis;insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65); islet cellantigen (ICA512; ICA12) with insulin dependent diabetes. An associationof GAD epitopes with diabetes is described in a number of publications,including U.S. Pat. No. 5,212,447; and European patent application no.94.927940.0. An association of insulin epitopes with autoimmuneinsulitis is described in Griffin et al. (1995) Am. J. Pathol.147:845-857. Rudy et al. (1995) Mol. Med. 1:625-633 disclose an epitopethat is similar in GAD and proinsulin.

[0026] The protein components of myelin proteins, including myelin basicprotein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein(MAG) and myelin oligodendrocyte glycoprotein (MOG), are of particularinterest for use as immunogens of the invention. The suppression of Tcell responsiveness to these antigens is used to prevent or treatdemyelinating diseases.

[0027] In one embodiment of the invention, the vaccine autoantigen isproteolipid. For convenience, a reference sequence of human PLP isprovided as SEQ ID NO:1; and human myelin basic protein as SEQ ID NO:3.Proteolipid is a major constituent of myelin, and is known to beinvolved in demyelinating diseases (see, for example Greer et al. (1992)J. Immunol. 149:783-788 and Nicholson (1997) Proc. Natl. Acad. Sci. USA94:9279-9284).

[0028] The integral membrane protein PLP is a dominant autoantigen ofmyelin. Determinants of PLP antigenicity have been identified in severalmouse strains, and include residues 139-151 (Tuohy et al. (1989) J.Immunol. 142:1523-1527), 103-116 (Tuohy et al. (1988) J. Immunol.141:1126-1130], 215-232 (Endoh et al. (1990) Int. Arch. Allergy Appl.Immunol. 92:433-438), 43-64 (Whitham et al. (1991) J. Immunol.147:3803-3808) and 178-191 (Greer, et al. (1992) J. Immunol.149:783-788). Immunization with native PLP or with synthetic peptidescorresponding to PLP epitopes induces EAE. Analogues of PLP peptidesgenerated by amino acid substitution can prevent EAE induction andprogression (Kuchroo et al. (1994) J. Immunol. 153:3326-3336, Nicholsonet al. (1997) Proc. Natl. Acad. Sci. USA 94:9279-9284).

[0029] MBP is an extrinsic myelin protein that has been studiedextensively. At least 26 MBP epitopes have been reported (Meinl et al.(1993) J. Clin. Invest. 92:2633-2643). Of particular interest for use inthe present invention are residues 1-11, 59-76 and 87-99. Analogues ofMBP peptides generated by truncation have been shown to reverse EAE(Karin et al. (1998) J. Immunol. 160:5188-5194). DNA encodingpolypeptide fragments may comprise coding sequences for immunogenicepitopes, e.g. myelin basic protein p84-102, more particularly myelinbasic protein p87-99, (SEQ ID NO:11) VHFFKNIVTPRTP (p87-99), or even thetruncated 7-mer peptide (SEQ ID NO:12) FKNIVTP. The sequences of myelinbasic protein exon 2, including the immunodominant epitope bordered byamino acids 59-85, are also of interest. For examples, see Sakai et al.(1988) J Neuroimmunol 19:21-32; Baxevanis et al (1989) J Neuroimmunol22:23-30; Ota et al (1990) Nature 346:183-187; Martin et al (1992) JImmunol. 148:1350-1366, Valli et al (1993) J Clin Inv 91:616. Theimmunodominant MBP(84-102) peptide has been found to bind with highaffinity to DRB1*1501 and DRB5*0101 molecules of the disease-associatedDR2 haplotype. Overlapping but distinct peptide segments were importantfor binding to these molecules; hydrophobic residues (Val189 and Phe92)in the MBP (88-95) segment for peptide binding to DRB1*1501 molecules;hydrophobic and charged residues (Phe92, Lys93) in the MBP (89-101/102)sequence contributed to DRB5*0101 binding.

[0030] The transmembrane glycoprotein MOG is a minor component of myelinthat has been shown to induce EAE. Immunodominant MOG epitopes that havebeen identified in several mouse strains include residues 1-22, 35-55,64-96 (deRosbo et al. (1998) J. Autoimmunity 11:287-299, deRosbo et al.(1995) Eur J Immunol. 25:985-993) and 41-60 (Leadbetter et al. (1998) JImmunol 161:504-512).

[0031] For the treatment of diabetes, immunogens of interest includeIA-2; IA-2beta; GAD; insulin; proinsulin; HSP; glima 38; ICA69; and p52.For example, insulin (which sequence is publicly available, for examplefrom Sures et al. (1980) Science 208:57-59; Bell et al. (1979) Nature282:525-527; and Bell et al. (1980) Nature 284:26-32) has been found tohave immunodominant epitopes in the B chain, e.g. residues 9-23; as wellas in the pre-proinsulin leader sequence. Other autoantigens associatedwith diabetes include glutamic acid decarboxylase 65 (GAD65), e.g.residues 206-220; 221-235, 286-300; 456-470; and peptides includingresidues p247, p509; p524 (Kauffman et al. (1993) Nature 366:69-72).

[0032] A DNA expression cassette encoding at least a portion of anautoantigen, usually as part of a vector, is introduced into tissue ofthe vaccine recipient. The minigene is expressed in the tissue, and theencoded polypeptide acts as an immunogen, or antigen. The autoantigensequence may be from any mammalian or avian species, e.g. primate sp.,particularly humans; rodents, including mice, rats and hamsters;rabbits; equines, bovines, canines, felines; etc. Of particular interestare the human and mouse autoantigen segments. Generally, the sequencewill have the same species of origin as the animal host, preferably itwill be autologous

[0033] The subject DNA expression cassette will comprise most or all ofthe sequence encoding an autoantigen fragment, as defined by Kabat etal, supra. The coding sequence may be truncated at the 5′ or 3′ terminusand may be a fragment of the complete polypeptide sequence. In oneembodiment of the invention, the sequence encodes a peptide fragmentthat is known to be presented to pathogenic T cells, for examplepeptides presented by Class II MHC molecules of the host. Such peptideshave been described in the literature, and are typically of about 8 toabout 30 amino acids in length.

[0034] The vaccine may be formulated with one or a cocktail ofautoantigen sequences, While it has been found that a single sequence iscapable of suppressing a response to multiple epitopes, it may bedesirable in some cases to include multiple sequences, where eachencodes a different epitope. For example, see Leadbetter et al. (1998)J. Immunol. 161:504-512. A formulation comprised of multiple codingsequences of distinct PLP epitopes may be used to induce a more potentand/or sustained suppressive response. By specifically targetingmultiple autoreactive T cell populations, such a formulation may slow orprevent the development of autoantigen resistance. The use of PLPsequences in combination with other myelin protein epitopes mayeffectively suppress the repertoire of myelin-reactive T cells. Similarautoantigen combinations to suppress autoimmune response, e.g., glutamicacid decarboxylase (GAD) and pancreatic islet cell autoantigen for thetreatment of insulin dependent diabetes, are contemplated.

[0035] In addition to the specific epitopes and polypeptides ofautoantigens, the immune response may be enhanced by the inclusion ofCpG sequences, as described by Krieg et al. (1998) Trends Microbiol.6:23-27, and helper sequence, King et al. (1998) Nat. Med. 4:1281-1286.Biological effects of DNA motifs like unmethylated CpG dinucleotides inparticular base contexts (CpG-S motifs) may modulate innate immuneresponses when injected to animals. Low numbers of CpG motifs, or thepresence of imperfect motifs, may act in the development of anergy byimmunization with autoantigens.

[0036] The polypeptide coding sequence, which may be autoantigen orcytokine, sequences are inserted into an appropriate expressioncassette. The expression construct is prepared in conventional ways. Thecassette will have the appropriate transcriptional and translationalregulatory sequences for expression of the sequence in the vaccinerecipient cells. The cassette will generally be a part of a vector,which contains a suitable origin of replication, and such genes encodingselectable markers as may be required for growth, amplification andmanipulation of the vector, prior to its introduction into therecipient. Suitable vectors include plasmids, YACs, BACs, bacteriophage,retrovirus, and the like. Conveniently, the expression vector will be aplasmid. Prior to vaccination, the cassette may be isolated from vectorsequences by cleavage, amplification, etc. as known in the art. Forinjection, the DNA may be supercoiled or linear, preferably supercoiled.The cassette may be maintained in the host cell for extended periods oftime, or may be transient, generally transient. Stable maintenance isachieved by the inclusion of sequences that provide for integrationand/or maintenance, e.g. retroviral vectors, EBV vectors and the like.

[0037] The expression cassette will generally employ an exogenoustranscriptional initiation region, i.e. a promoter other than thepromoter which is associated with the T cell receptor in the normallyoccurring chromosome. The promoter is functional in host cells,particularly host cells targeted by the cassette. The promoter may beintroduced by recombinant methods in vitro, or as the result ofhomologous integration of the sequence by a suitable host cell. Thepromoter is operably linked to the coding sequence of the autoantigen toproduce a translatable mRNA transcript. Expression vectors convenientlywill have restriction sites located near the promoter sequence tofacilitate the insertion of autoantigen sequences.

[0038] Expression cassettes are prepared comprising a transcriptioninitiation region, which may be constitutive or inducible, the geneencoding the autoantigen sequence, and a transcriptional terminationregion. The expression cassettes may be introduced into a variety ofvectors. Promoters of interest may be inducible or constitutive, usuallyconstitutive, and will provide for high levels of transcription in thevaccine recipient cells. The promoter may be active only in therecipient cell type, or may be broadly active in many different celltypes. Many strong promoters for mammalian cells are known in the art,including the β-actin promoter, SV40 early and late promoters,immunoglobulin promoter, human cytomegalovirus promoter, retroviralLTRs, etc. The promoters may or may not be associated with enhancers,where the enhancers may be naturally associated with the particularpromoter or associated with a different promoter.

[0039] A termination region is provided 3′ to the coding region, wherethe termination region may be naturally associated with the variableregion domain or may be derived from a different source. A wide varietyof termination regions may be employed without adversely affectingexpression.

[0040] The various manipulations may be carried out in vitro or may beperformed in an appropriate host, e.g. E. coli. After each manipulation,the resulting construct may be cloned, the vector isolated, and the DNAscreened or sequenced to ensure the correctness of the construct. Thesequence may be screened by restriction analysis, sequencing, or thelike.

[0041] A small number of nucleotides may be inserted at the terminus ofthe autoantigen sequence, usually not more than 20, more usually notmore than 15. The deletion or insertion of nucleotides will usually beas a result of the needs of the construction, providing for convenientrestriction sites, addition of processing signals, ease of manipulation,improvement in levels of expression, or the like. In addition, one maywish to substitute one or more amino acids with a different amino acidfor similar reasons, usually not substituting more than about five aminoacids in the region.

[0042] In one embodiment of the invention the autoantigen isco-vaccinated with DNA sequences encoding a Th2 cytokine, which groupincludes IL-4, IL-10, TGF-β, etc. IL4 is of particular interest. Thelymphokine IL-4 has T-cell and mast cell growth factor activities. HumanIL4 is an 18-kD glycoprotein. For convenience the amino acid sequence isprovided herein as SEQ ID NO:13, and the DNA sequence as SEQ ID NO:14(Yokota et al. (1986) P.N.A.S. 83:5894-5898). This sequence is thepreferred sequence of the invention. However, the invention is notlimited to the use of this sequence in constructs of the invention. Alsoof use are closely related variant sequences that have the samebiological activity, or substantially similar biological activity. Aspecific STAT6 DNA-binding target site is found in the promoter of theIL4 receptor gene; and STAT6 activates IL4 gene expression via thissite. Interferons inhibit IL4-induced activation of STAT6 andSTAT6-dependent gene expression, at least in part, by inducingexpression of SOCS1 (see Kotanides et al. (1996) J. Biol. Chem.271:25555-25561).

[0043] Variant sequences encode protein subunits which, when present ina DNA construct of the invention, give the protein one or more of thebiological properties of IL-4 as described above. DNA sequences of theinvention may differ from a native IL-4 sequence by the deletion,insertion or substitution of one or more nucleotides, provided that theyencode a protein with the appropriate biological activity as describedabove. Similarly, they may be truncated or extended by one or morenucleotides. Alternatively, DNA sequences suitable for the practice ofthe invention may be degenerate sequences that encode the naturallyoccurring IL-4 protein. Typically, DNA sequences of the invention haveat least 70%, at least 80%, at least 90%, at least 95% or at least 99%sequence identity to a native IL-4 coding sequence. They may originatefrom any species, though DNAs encoding human proteins are preferred.Variant sequences may be prepared by any suitable means known in theart.

[0044] With respect of substitutions, conservative substitutions arepreferred. Typically, conservative substitutions are substitutions inwhich the substituted amino acid is of a similar nature to the onepresent in the naturally occurring protein, for example in terms ofcharge and/or size and/or polarity and/or hydrophobicity. Similarly,conservative substitutions typically have little or no effect on theactivity of the protein. Proteins of the invention that differ insequence from naturally occurring IL-4 may be engineered to differ inactivity from naturally occurring IL-4. Such manipulations willtypically be carried out at the nucleic acid level using recombinanttechniques, as known in the art.

[0045] The vaccine may be formulated with one or a cocktail ofautoantigen sequences, which may be on the same or different vectors.The DNA vectors are suspended in a physiologically acceptable buffer,generally an aqueous solution e.g. normal saline, phosphate bufferedsaline, water, etc. Stabilizing agents, wetting and emulsifying agents,salts for varying the osmotic pressure or buffers for securing anadequate pH value, and skin penetration enhancers can be used asauxiliary agents. The DNA will usually be present at a concentration ofat least about 1 ng/ml and not more than about 10 mg/ml, usually atabout from 100 μg to 1 mg/ml.

[0046] In some embodiments of the the present invention, the patient isadministed both an autoantigen encoding sequence and a Th2 cytokineencoding sequence. The cytokine and autoantigen can be deliveredsimultaneously, or within a short period of time, by the same or bydifferent routes. In one embodiment of the invention, the two sequencesare co-formulated, meaning that they are delivered together as part of asingle composition. The coding sequences may be associated with oneanother by covalent linkage in a single nucleic acid molecule, wherethey may be present as two distinct coding sequences separated by atranslational stop, or may be be present as a single fusion protein. Thetwo sequences may also by joined by non-covalent interaction such ashydrophobic interaction, hydrogen bonding, ionic interaction, van derWaals interaction, magnetic interaction, or combinations thereof.Alternatively, the two constructs may simply be mixed in a commonsuspension, or encapsulated together in some form of delivery devicesuch as, for example, an alginate device, a liposome, chitosan vesicle,etc. (see, for example, WO 98/33520, incorporated herein by reference).

[0047] The vaccine may be fractionated into two or more doses, of atleast about 1 μg, more usually at least about 100 μg, and preferably atleast about 1 mg per dose, administered from about 4 days to one weekapart. In some embodiments of the invention, the individual is subjectto a series of vaccinations to produce a full, broad immune response.According to this method, at least two and preferably four injectionsare given over a period of time. The period of time between injectionsmay include from 24 hours apart to two weeks or longer betweeninjections, preferably one week apart. Alternatively, at least two andup to four separate injections are given simultaneously at differentparts of the body.

[0048] The DNA vaccine is injected into muscle or other tissuesubcutaneously, intradermally, intravenously, orally or directly intothe spinal fluid. Of particular interest is injection into skeletalmuscle. The genetic vaccine may be administered directly into theindividual to be immunized or ex vivo into removed cells of theindividual which are reimplanted after administration. By either route,the genetic material is introduced into cells which are present in thebody of the individual. Alternatively, the genetic vaccine may beintroduced by various means into cells that are removed from theindividual. Such means include, for example, transfection,electroporation and microprojectile bombardment. After the geneticconstruct is taken up by the cells, they are reimplanted into theindividual. Otherwise non-immunogenic cells that have genetic constructsincorporated therein can betaken from one individual and implanted intoanother.

[0049] An example of intramuscular injection may be found in Wolff etal. (1990) Science 247:1465-1468. Jet injection may also be used forintramuscular administration, as described by Furth et al. (1992) AnalBiochem 205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”.Microparticle DNA vaccination has been described in the literature (see,for example, Tang et al. (1992) Nature 356:152-154). Goldmicroprojectiles are coated with the vaccine cassette, then bombardedinto skin cells.

[0050] The genetic vaccines are formulated according to the mode ofadministration to be used. One having ordinary skill in the art canreadily formulate a genetic vaccine that comprises a genetic construct.In cases where intramuscular injection is the chosen mode ofadministration, an isotonic formulation is used. Generally, additivesfor isotonicity can include sodium chloride, dextrose, mannitol,sorbitol and lactose. Isotonic solutions such as phosphate bufferedsaline are preferred. Stabilizers include gelatin and albumin.

[0051] According to the present invention, prior to or contemporaneouslywith administration of the genetic construct, cells may be administereda cell stimulating or cell proliferative agent, which terms are usedinterchangeably and refer to compounds that stimulate cell division andfacilitate DNA and RNA uptake.

[0052] Bupivacaine or compounds having a functional similarity may beadministered prior to or contemporaneously with the vaccine. Bupivacaineis a homologue of mepivacaine and related to lidocaine. It rendersmuscle tissue voltage sensitive to sodium challenge and effects ionconcentration within the cells. In addition to bupivacaine, mepivacaine,lidocaine and other similarly acting compounds, other contemplated cellstimulating agents include lectins, growth factors, cytokines andlymphokines such as platelet derived growth factor (PDGF), gCSF, gMCSF,epidermal growth factor (EGF) and IL4. About 50 μl to about 2 ml of 0.5%bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceuticalcarrier may be administered to the site where the vaccine is to beadministered, preferably, 50 .mu.l to about 1500 μl, more preferablyabout 1 ml. The genetic vaccine may also be combined with collagen as anemulsion and delivered intraperatonally. The collagen emulsion providesa means for sustained release of DNA. 50 μl to 2 ml of collagen areused.

[0053] The efficiency of DNA vaccination may be improved by injection ofcardiotoxin into the tissue about one week prior to the vaccination, asdescribed by Davis et al. (1993) FEBS Lett. 333:146-150, and in theexamples. The cardiotoxin stimulates muscle degeneration andregeneration. The muscle is injected with from about 0.1 to 10 μM ofcardiotoxin dissolved in a pharmacologically acceptable vehicle.

[0054] The condition that is being treated, and the host immune statuswill determine the choice of autoantigen sequence(s). The host may beassessed for immune responsiveness to a candidate vaccine autoantigen byvarious methods known in the art.

[0055] The diagnosis may determine the level of reactivity, e.g. basedon the number of reactive T cells found in a sample, as compared to anegative control from a naive host, or standardized to a data curveobtained from one or more patients. In addition to detecting thequalitative and quantitative presence of auto-antigen reactive T cells,the T cells may be typed as to the expression of cytokines known toincrease or suppress inflammatory responses. It may also be desirable totype the epitopic specificity of the reactive T cells.

[0056] T cells may be isolated from patient peripheral blood, lymphnodes, or preferably from the site inflammation. Reactivity assays maybe performed on primary T cells, or the cells may be fused to generatehybridomas. Such reactive T cells may also be used for further analysisof disease progression, by monitoring their in situ location, T cellreceptor utilization, etc. Assays for monitoring T cell responsivenessare known in the art, and include proliferation assays and cytokinerelease assays.

[0057] Proliferation assays measure the level of T cell proliferation inresponse to a specific antigen, and are widely used in the art. In anexemplary assay, patient lymph node, blood or spleen cells are obtained.A suspension of from about 10⁴ to 10⁷ cells, usually from about 10⁵ to10⁶ cells is prepared and washed, then cultured in the presence of acontrol antigen, and test antigens. The test antigens may be peptides ofany autologous antigens suspected of inducing an inflammatory T cellresponse. The cells are usually cultured for several days.Antigen-induced proliferation is assessed by the monitoring thesynthesis of DNA by the cultures, e.g. incorporation of ³H-thymidineduring the last 18 H of culture.

[0058] Enzyme linked immunosorbent assay (ELISA) assays are used todetermine the cytokine profile of reactive T cells, and may be used tomonitor for the expression of such cytokines as IL-2, IL-4, IL-5, γIFN,etc. The capture antibodies may be any antibody specific for a cytokineof interest, where supernatants from the T cell proliferation assays, asdescribed above, are conveniently used as a source of antigen. Afterblocking and washing, labeled detector antibodies are added, and theconcentrations of protein present determined as a function of the labelthat is bound.

[0059] The above diagnostic assays may be performed with variouspeptides derived from the autologous protein of interest. A series ofpeptides having the sequence of an auto-antigen, e.g. PLP, MBP, etc. maybe used. Possible peptides may be screened to determine which areimmunodominant in the context of autoimmune disease.

[0060] The immunodominant peptides may be defined by screening with apanel of peptides derived from the test protein. The peptides have theamino acid sequence of a portion of the protein, usually at least about8 and not more than about 30 amino acids, more usually not more thanabout 20 amino acids in length. The panel of peptides will represent thelength of the protein sequence, i.e. all residues are present in atleast one peptide. Preferably overlapping peptides are generated, whereeach peptide is frameshifted from 1 to 5 amino acids, thereby generatinga more complete set of epitopes. The peptides may be initially screenedin pools, and later screened for the exact epitope to which the T cellwill respond, as previously described. Immunodominant peptides arerecognized by a significant fraction of the HLA restricted, responsivehybridomas, usually at least about 10%, more usually at least about 25%,and may be as much as 80%.

[0061] The subject therapy will desirably be administered during thepresymptomatic or preclinical stage of the disease, and in some casesduring the symptomatic stage of the disease. Early treatment ispreferable, in order to prevent the loss of function associated withinflammatory tissue damage. The presymptomatic, or preclinical stagewill be defined as that period not later than when there is T cellinvolvement at the site of disease, e.g. islets of Langerhans, synovialtissue, thyroid gland, etc., but the loss of function is not yet severeenough to produce the clinical symptoms indicative of overt disease. Tcell involvement may be evidenced by the presence of elevated numbers ofT cells at the site of disease, the presence of T cells specific forautoantigens, the release of performs and granzymes at the site ofdisease, response to immunosuppressive therapy, etc.

[0062] Degenerative joint diseases may be inflammatory, as withseronegative spondylarthropathies, e.g. ankylosing spondylitis andreactive arthritis; rheumatoid arthritis; gout; and systemic lupuserythematosus. The degenerative joint diseases have a common feature, inthat the cartilage of the joint is eroded, eventually exposing the bonesurface. Destruction of cartilage begins with the degradation ofproteoglycan, mediated by enzymes such as stromelysin and collagenase,resulting in the loss of the ability to resist compressive stress.Alterations in the expression of adhesion molecules, such as CD44(Swissprot P22511), ICAM-1 (Swissprot P05362), and extracellular matrixprotein, such as fibronectin and tenascin, follow. Eventually fibrouscollagens are attacked by metalloproteases, and when the collagenousmicroskeleton is lost, repair by regeneration is impossible.

[0063] There is significant immunological activity within the synoviumduring the course of inflammatory arthritis. While treatment duringearly stages is desirable, the adverse symptoms of the disease may be atleast partially alleviated by treatment during later stages. Clinicalindices for the severity of arthritis include pain, swelling, fatigueand morning stiffness, and may be quantitatively monitored by Pannuscriteria. Disease progression in animal models may be followed bymeasurement of affected joint inflammation. Therapy for inflammatoryarthritis may combine the subject treatment with conventional NSAIDtreatment. Generally, the subject treatment will not be combined withsuch disease modifying drugs as cyclosporin A, methotrexate, and thelike.

[0064] A quantitative increase in myelin autoreactive T cells with thecapacity to secrete IFN-gamma is associated with the pathogenesis of MSand EAE, suggesting that autoimmune inducer/helper T lymphocytes in theperipheral blood of MS patients may initiate and/or regulate thedemyelination process in patients with MS. The overt disease isassociated with muscle weakness, loss of abdominal reflexes, visualdefects and paresthesias. During the presymptomatic period there isinfiltration of leukocytes into the cerebrospinal fluid, inflammationand demyelination. Family histories and the presence of the HLAhaplotype DRB1*1501, DQA1*0102, DQB1*0602 are indicative of asusceptibility to the disease. Markers that may be monitored for diseaseprogression are the presence of antibodies in the cerebrospinal fluid,“evoked potentials” seen by electroencephalography in the visual cortexand brainstem, and the presence of spinal cord defects by MRI orcomputerized tomography. Treatment during the early stages of thedisease will slow down or arrest the further loss of neural function.

[0065] Human insulin-dependent diabetes mellitus (IDDM) is a diseasecharacterized by autoimmune destruction of the p cells in the pancreaticislets of Langerhans. An animal model for the disease is the non-obesediabetic (NOD) mouse, which develops autoimmunity. NOD micespontaneously develop inflammation of the islets and destruction of theβ cells, which leads to hyperglycemia and overt diabetes. Both CD4⁺ andCD8⁺ T cells are required for diabetes to develop: CD4⁺ T cells appearto be required for initiation of insulitis, cytokine-mediateddestruction of p cells, and probably for activation of CD8⁺ T cells. TheCD8⁺ T cells in turn mediate β cell destruction by cytotoxic effectssuch as release of granzymes, perforin, TNFα and IFNγ. Reactivities toseveral candidate autoantigens, including epitopes of insulin andglutamic acid decarboxylase (GAD), have been detected.

[0066] In one embodiment of the invention, the coding sequence used forvaccination provides for an immunogenic insulin epitope. Immunodominantepitopes include the B chain, in particular residues 9-23, which havebeen implicated in both human disease and in animal models. Epitopes ofthe pre-proinsulin have also been implicated as immunodominant epitopes.Protection from diabetes is associated with down regulation of IFN-γ andIL-10 in pancreatic lymph node cells in response to the insulin peptideencoded in the vaccine. It has been found that T cells immunized with animmunodominant insulin epitope express substantially lower levels ofIFN-γ in response to activation.

[0067] The depletion of β cells results in an inability to regulatelevels of glucose in the blood. Overt diabetes occurs when the level ofglucose in the blood rises above a specific level, usually about 250mg/dl. In humans a long presymptomatic period precedes the onset ofdiabetes. During this period there is a gradual loss of pancreatic βcell function. The disease progression may be monitored in individualsdiagnosed by family history and genetic analysis as being susceptible.The most important genetic effect is seen with genes of the majorhistocompatibility locus (IDDM1), although other loci, including theinsulin gene region (IDDM2) also show linkage to the disease (see Davieset al, supra and Kennedy et al. (1995) Nature Genetics 9:293-298).

[0068] Markers that may be evaluated during the presymptomatic stage arethe presence of insulitis in the pancreas, the level and frequency ofislet cell antibodies, islet cell surface antibodies, aberrantexpression of Class II MHC molecules on pancreatic β cells, glucoseconcentration in the blood, and the plasma concentration of insulin. Anincrease in the number of T lymphocytes in the pancreas, islet cellantibodies and blood glucose is indicative of the disease, as is adecrease in insulin concentration. After the onset of overt diabetes,patients with residual b cell function, evidenced by the plasmapersistence of insulin C-peptide, may also benefit from the subjecttreatment, to prevent further loss of function.

[0069] Mammalian species susceptible to inflammatory conditions includecanines and felines; equines; bovines; ovines; etc. and primates,particularly humans. Animal models, particularly small mammals, e.g.murine, lagomorpha, etc. may be used for experimental investigations.Animal models of interest include those involved with the production ofantibodies having isotypes associated with IL-4 production, e.g. IgE,IgG1 and IgG4. Other uses include investigations where it is desirableto investigate a specific effect in the absence of T cell mediatedinflammation.

[0070] It is to be understood that this invention is not limited to theparticular methodology, protocols, formulations and reagents described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0071] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a complex” includes a plurality of such complexes and reference to “theformulation” includes reference to one or more formulations andequivalents thereof known to those skilled in the art, and so forth.

[0072] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0073] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, themethods and methodologies that are described in the publications whichmight be used in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

[0074] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention. Efforts have been madeto ensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight, andpressure is at or near atmospheric.

Experimental

[0075] Materials and Methods

[0076] Animals. Six to eight week old female SJL/J mice were purchasedfrom Jackson Laboratory (Bar Harbor, Me.).

[0077] Antigens. Peptides were synthesized on a peptide synthesizer(model 9050: MilliGen, Burlington, Mass.) by standard9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC.Structure was confirmed by amino acid analysis and mass spectroscopy.Peptides used for the experiments were: PLP139-151 (SEQ ID NO:5HSLGKWLGHPDKF), PLP139-151 L144/R147 (SEQ ID NO:6 HSLGKLLGRPDKF), andPLP178-191 (SEQ ID NO:7 NTWTTCQSIAFPSK). Guinea pig spinal cordhomogenate (SCH) was used after lyophilization.

[0078] PLP peptide expression vector. Three minigenes, each one encodinga PLP epitope, were constructed by annealing two oligonucleotides with a16 mer overlapping complementary sequence (underlined), and extendingwith DNA polymerase and dNTPs: PLP (178-191): SEQ ID NO:8 5′-CTGGAGACCAGAATACCTGG ACCACCTGCC AGTCTATTGC CTTCCCTAGC AAGTCTAGAT AGCTA-3′ PLP(139-151): SEQ ID NO:9 5′-CTCGAGACCA TGCATTGTTT GGGAAAATGGCTAGGACATCCCGACAAGTTTTCTAGATAGCTA -3′. PLP (139-151) L144/R147 SEQ IDNO:10: 5′-CTCGAGACCATGCATTGTTTGGGAAAACTACTAGGACGCCCCGACAAGTTTTCTAGATAGCTA -3′.

[0079] These oligonucleotide duplexes were designed to incorporate Xho Iand Xba I restriction sites.

[0080] The products were cloned into the multiple cloning region ofpTARGET Vector (Promega, Madison, Wis.), a mammalian expression vectordriven by the CMV promoter. Positive clones were identified by colorscreening and correct orientation of the inserts was confirmed by DNAautomatic sequencing. Purification of the plasmid DNA was done by Wizardplus Maxipreps (Promega) according to manufacturer instructions.

[0081] DNA immunization protocol. Experimental animals were injected inthe left quadriceps with 0.1 ml of 0.25% bupivacaine-HCl (Sigma, St.Louis, Mo.) in PBS. Two and ten days later, mice were injected with 0.05ml of plasmid DNA (1 mg/ml in PBS), in the same muscle.

[0082] ELISA for anti-PLP139-151 or anti-guinea pig SCH antibody titers.Polystyrene 96 well microtiter plates (Dynatech, Chantilly, Va.) werecoated with 0.1 ml of either peptide or guinea pig SCH, diluted in PBSat a concentration of 0.01 mg/ml in PBS. After blocking with PBS+0.5%fetal calf serum (Gibco) and 0.05% tween 20 (Bio-Rad, Hercules, Calif.),mouse sera were incubated for two hours at room temperature and antibodybinding was tested by the addition of alkaline phosphatase-conjugatedgoat anti-mouse IgG (Southern Biotechnology, Birmingham, Ala.). Afterthe addition of the enzyme substrate, plates were read at 405 nm in anELISA reader. FIG. 1 shows the results for sera taken seven days afterthe second intramuscular injection expressed as O.D. of individualsamples in a group of ten animals. O.D. values for preimmune sera were:dilution 1:10:0.12, dilution 1:20:0.08, and dilution 1:40:0.03.

[0083] EAE induction. PLP139-151 peptide was dissolved in PBS to aconcentration of 2 mg/ml and emulsified with an equal volume ofIncomplete Freund's Adjuvant supplemented with 4 mg/ml heat-killedmycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, Mich.).Mice were injected subcutaneously with 0.1 ml of the peptide emulsionand, on the same day and 48 h later, intravenously with 0.1 ml of 4μg/ml Bordetella Pertussis toxin in PBS. Experimental animals werescored as follows: 0=no clinical disease; 1=tail weakness or paralysis;2=hind limb weakness; 3=hind limb paralysis; 4=forelimb weakness orparalysis; 5=moribund or dead animal.

[0084] Lymph node cell proliferation assays. Draining lymph nodes wereremoved from mice after the acute phase of disease and lymph node cells(LNC) were tested in vitro for specific proliferative responses to thePLP139-151 peptide. Cultures were prepared in flat bottom 96 wellmicrotiter plates in a volume of 0.2 ml/well at a cell concentration of2.5×10⁶/ml. The tissue culture media for the assay consisted of RPMI1640 supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), penicillin (100 U/ml), streptomycin (0.1mg/ml), 2-mercaptoethanol (5×10⁻⁵ M), and 1% autologous fresh normalmouse serum. After 72 h of incubation at 37° C., cells were pulsed for18 h with 1 μCi/well of (³H)thymidine. Plates were harvested and(³H)thymidine incorporation was measured in a scintillation counter.After recovery from the acute phase of disease animals injected eitherwith DNA coding for PLP139-151 or control vector, pTARGET weresacrified, and draining LNC were isolated. Cells were tested in vitro bystimulation with different concentrations of the peptide PLP139-151 orthe control peptide PLP178-191. Proliferative responses from pooled LNCof groups of five animals are shown in FIG. 2 as mean CPM±SD oftriplicate wells. CPM of Concanavalin A (0.001 mg/ml) stimulated LNCwere 102401 for group A and 76702 for group B.

[0085] Cytokine determination. Draining LNC (10⁷ cells/ml) fromexperimental animals were taken after the acute phase of the disease andstimulated in vitro with varying concentrations of antigen. After 24 and48 h of stimulation supernatants were collected and tested by sandwichELISA.

[0086] Ribonuclease protection assay. For mRNA detection tissue RNAsamples of LNC from experimental animals were tested using theMulti-Probe RNase Protection Assay (RPA) System, RiboQuant (Pharmigen,San Diego, Calif.) according to manufacturer instructions.

[0087] Fluorocytometric analysis. Spleen cells (5×10⁶/ml) from naiveSJL/J mice were incubated in the presence of plasmid DNA coding for thePLP139-151 sequence (0.01 mg/ml) at 37° C. After 24 h cells werecollected and analyzed on FACScan flow cytometer (Becton Dickinson). Thefollowing antibody conjugates were used: FITC anti-mouse CD80, clone16-10A1; FITC anti-mouse CD86, clone GL1; FITC anti-mouse I-A^(k), clone10-3.6; R-PE conjugated anti-mouse B220, clone RA3-6B2; R-PE conjugatedanti-mouse CD11b, clone M1/70; PE conjugated anti-mouse, clone GK 1.5.All antibodies were purchased from Pharmigen, San Diego, Calif. After 24h of in vitro incubation without DNA (non) or with plasmid DNA codingfor the PLP139-151 peptide [DNA (PLP139-151)], spleen cells were stainedwith anti-Mac 1 mAb, anti-B220 mAb, anti-B7.2 mAb, and anti-B7.2 mAb asindicated. Blank refers to nonspecific background staining. Resultsshown in FIG. 4 are representative of three experiments.

[0088] Results

[0089] The minigene, coding for the PLP139-151 peptide, was cloned intoan expression vector and injected intramuscularly into SJL/J mice,twice, at one week intervals. Ten days after the last injection,experimental animals were bled and their sera were tested for thepresence of specific antibodies. As shown in FIG. 1, anti-PLP139-151 IgGtiters can be detected in the mice previously injected with thePLP139-151 minigene. Thus, specific serological immune responses areinduced with this particular construct.

[0090] To determine whether injection of DNA containing PLP sequencescan be effective in protecting mice from EAE induction, the PLP139-151minigene construct was injected, intramuscularly, twice, at one weekintervals. Ten days after the last injection, mice were challenged withthe PLP139-151 peptide emulsified in CFA. As shown in Table 1,amelioration of acute clinical disease is observed in the animalsvaccinated with the PLP139-151 plasmid vector, as compared with thecontrol plasmid group. Onset of disease was delayed compared to thecontrol plasmid group (11.5±0.5 days, p<0.008), mean peak diseaseseverity was reduced (p<0.005), and mean disease score was reduced(p<0.0005). In addition, other groups were injected with either a) aplasmid containing a minigene encoding the altered peptide ligand PLPp139-151 (W144>L, H147>R), b) a plasmid containing a minigene encodingthe PLP epitope p178-191. Onset of disease was delayed (11.6±0.5 days,p<0.009) and mean peak disease score was reduced (p<0.02) with theminigene encoding the altered peptide ligand (W144, H147). Also, onsetof disease was delayed (11.5±0.4 days, p<0.003), mean peak diseaseseverity was reduced (p<0.007), and mean disease score was reduced(p<0.0001) with the minigene encoding the PLP peptide p178-191. TABLE 1EAE induction in DNA immunized SJL/J mice. Mean disease Mean peak DNAPercent score on Mean day of disease injected incidence day 11^(†)disease onset severity PLP 68 (13/19)* 0.9 ± 0.3 11.5 ± 0.5 1.7 ± 0.4139-151 (p < 0.0005)^(¶) (p < 0.008) (p < 0.005) PLP 70 (14/20) 0.6 ±0.2 11.5 ± 0.4 1.8 ± 0.3 178-191 (p < 0.0001) (p < 0.0035) (p < 0.007)PLP 85 (17/20) 1.2 ± 0.3 11.6 ± 0.5 2.0 ± 0.3 139-151 (p < 0.001) (p <0.009) (p < 0.01) (L > R) pTAR- 90 (18/20) 2.7 ± 0.3  10.1 ± 0.27 3.1 ±0.3 GET Non- 100 (10/10) 2.1 ± 0.7 9.9 ± 0.4 3.3 ± 0.3 plasmid

[0091] Mice, injected with DNA and further challenged with theencephalitogenic peptide PLP139-151, were sacrificed after resolution ofthe acute phase of the clinical disease. Draining LNC were restimulatedin vitro with the PLP139-151 peptide and tested for their proliferativeresponses and cytokine production. FIG. 2 shows that LNC from miceinjected with DNA coding for the PLP139-151 peptide had lowerproliferative responses when compared with the LNC from control animals(p<0.01). FIG. 3 (A) shows that, when stimulated with the PLP139-151,LNC from mice immunized with the plasmid DNA coding for the PLP139-151region secrete lower levels of IL-2 and γ-interferon in comparison withcontrol groups. In order to assess levels of cytokine mRNA transcriptsin inflamed brain we utilized a ribonuclease protection assay on mRNAisolated from brain tissue. FIG. 3 (B) reveals a reduction in mRNAlevels of γ-interferon and IL-15 in mice immunized with the minigeneencoding the PLP139-151 region. Therefore, a correlation between lowincidence of clinical disease, reduced cellular responses, and lowlevels of IL-2, IL-15 and γ-interferon is evident in the PLP139-151 DNAvaccinated mice. The relative expression levels of cytokine mRNA's bandsshown in FIG. 3B were measured by densitometry. In order to correct forloading differences, the values were normalized according to the levelof expression of the housekeeping gene, GAPDH, within each sample. Thereis a reduction of expression level of the tested cytokines in brains ofmice vaccinated with the plasmid DNA coding for the PLP139-151determinant compared to pTARGET and PLP139-151 (L/R) plasmid DNAvaccinated mice.

[0092] In order to elucidate a mechanism for decreased T cell responses,we tested in vitro the effect of APCs, cultured in the presence of DNA,on the proliferative responses of PLP139-151 specific T cells.Splenocytes were incubated either with plasmid DNA coding for thePLP139-151 segment, or with the PLP139-151 peptide and used as a sourceof APC to stimulate L139 cells, a PLP139-151 specific T cell line.Proliferative responses of the L139 T cell line to the above APCs werecompared in the presence or absence of anti-CD28 antibody coated beads.As shown in Table 2, L139 cells responded to syngeneic APCs preincubatedwith the synthetic peptide PLP139-151 [8512 mean cpm]. This response isincreased with addition of anti-CD28 antibodies [127281 mean cpm].However, when the APCs were incubated with the plasmid DNA containingPLP139-151 coding sequence, L139 cells were unable to respond to APCs[3358 mean cpm], even in the presence of anti-CD28 antibodies [4532 meancpm]. This downregulation was not an effect of the plasmid itself, sinceAPCs incubated with plasmid containing an irrelevant sequence did notaffect the proliferative response of L139 cells to anti-CD28 antibodies[4532 cpm versus 26363 mean cpm, p<0.0001]. Therefore, PLP139-151specific T cells are unable to respond to CD28 co-stimulation whencultured in the presence of APC loaded with plasmid DNA coding for thePLP139-151 sequence. TABLE 2 Proliferative responses of PLP139-151specific T cells line in the presence of syngeneic splenocytes loadedwith either plasmid DNA or synthetic peptide. pTAR- PLP139- GET 151 HSV-PLP139- Anti-CD28 plasmid plasmid VP16 151 co- CPM³ DNA¹ DNA¹ peptide¹peptide¹ stimulation² (mean) − − − − −  2186 − − + − −  2402 − − + − + 15139 − − − + −  8512 − − − + + 127281 + − − − −  2331 + − − − + 26363* − + − − −  3358 − + − − +  4532* # peptide-specific T cell line,L139, were added to each well. After 48 hrs of further incubation platewas labeled with ³H-thymidine and proliferation was assessed byharvesting 18 later and counting ³H-thymidine incorporation. Todemonstrate that the exogenously applied naked DNA is taken up by thesplenocytes, and is expressed we used reverse transcriptase-polymerasechain reaction (RT-PCR) technique. Total RNA was purified from thesplenocytes # using the Rneasy total RNA kit (Quiagen Inc., Valencia,CA). RT-PCR was performed using the Access RT-PCR System (Promega Corp.,Madison, WI) and oligonucleotide primers specific for the PLP139-151minigene. Vector specific primers were used in a separate RT-PCRreaction to exclude the possibility of DNA contamination. A single bandcorresponding to the PLP139-151 minigene was amplified from total RNApurified from splenocytes loaded with the PLP139-151 plasmid DNA (datanot shown). # 37° C. Beads were washed extensively with PBS andresuspended in RPMI-10% FCS and allowed to block for at least 30 minutesat room temperature.

[0093] The present study demonstrates protection from immunization withplasmid DNA encoding myelin minigenes. A DNA vaccine was created byinsertion of the coding sequence for the PLP139-151 region into abacterial plasmid under the control of CMV promoter. This vector wasinjected into SJL/J mice prior to the induction of EAE by immunizationwith the PLP139-151 peptide in CFA. Animals receiving the plasmid codingfor the encephalitogenic epitope were protected from EAE induction.Analysis of the immune responses in protected animals demonstrates lowerT cell proliferation and decreased pro-inflammatory cytokine secretion,both in lymphoid organs and within the target organ, the brain, incomparison with the control group. These features suggest that DNAimmunization anergizes pathogenic T cells.

[0094] The ability of myelin minigene constructs to downregulate theco-stimulatory effect of anti-CD28 antibodies on a PLP-specific T cellline emphasizes its capacity to modulate APC-T cell interactions.Fluorocytometric analyses were carried out to determine whether DNAimmunization influences the surface expression of CD28 ligands on APCs.After 24 h of incubation with the plasmid DNA, splenocytes were stainedwith either anti-B7.1 (CD80) or anti-B7.2 (CD86) antibodies. As shown inFIG. 4, up regulation of B7.1 and B7.2 is observed in Mac-1 positivecells, but not in B220⁺ cells where downregulation of B7.2 was observed.I-A^(s) expression in spleen cells also increased in both Mac-1 and B220positive cells upon incubation with DNA.

[0095] Similar up-regulation of costimulatory molecules has beenobserved in vivo in peripheral blood lymphocytes and spleen cells ofanimals inoculated with DNA expression cassettes coding for the HIV coreprotein 55. In contrast to this observation we found that in autoimmuneresponses to PLP139-151 the changes of expression of co-stimulatorymolecules after DNA immunization exert a protective effect by modulationof the proliferative potential and cytokine production of autoreactiveT-cells. Recently it has been reported that in EAE, there is enhancementof B7.1 expression relative to B7.2 in the splenic environment, afinding that can help explain how the immune system tilts towardautoimmunity, rather than immunological ignorance of self. InterestinglyB7.2 increases in the CNS during active EAE and during relapses.Downregulation of B7.2 correlates with remission. Changes in B7-1 andB7-2 expression upon uptake of DNA by antigen presenting cells could bea key factor in regulating T-cell responses toward self-antigens inautoimmune diseases.

[0096] DNA vaccines have been effective in generating protective immuneresponses in several models of cancer, and of viral, bacterial, andparasitic infections. Although generation of Th1-like responses may be aproperty of DNA vaccines targeting non-self antigens, Th1 responseselicited to self with DNA vaccination have not been achieved.

[0097] Biological effects of DNA motifs like unmethylated CpGdinucleotides in particular base contexts (CpG-S motifs) may modulateinnate immune responses when injected to animals (Krieg, A. M. et al.1998. Trends in Microbiol. 6, 23-27). Although we cannot discard apossible effect of such sequences in the PLP 139-151 and PLP 139/151(L/R) constructs, the CG motifs in these inserts do not fulfill thecomplete criteria for a CpG-S motif.

[0098] Suppression of EAE has been reported in Lewis rats by previousimmunization with DNA encoding an immunodominant MBP peptide in tandemwith IgG Fc receptor. Vaccination suppressed clinical andhistopathological signs of EAE, and reduced the interferon γ productionafter challenge with MBP 68-85 peptide [Lobell et al. (1998) J. Exp.Med. 187:1543-1548]. Vaccination was unsuccessful without inclusion ofthe tandem IgG Fc construct. In the experiments presented here, therewas apparently no need for any tandem construct in conjunction with themyelin minigene. In both the present paper and in the experimentsutilizing DNA with the Fc IgG construct, defective Th1 immunity to selfwas observed. In contrast, our laboratory has reported induction ofprotective Th2-type responses by DNA immunization in EAE [Waisman, 1996supra.]. Therefore, the immune response to a DNA vaccine encoding selfmight be very different from what is observed with DNA vaccination toforeign antigens. It might be predicted that immune responses induced byself antigens encoded in DNA vaccines will parallel what has beenobserved for immunization with the same self-antigen in peptide orprotein form. Our results suggest that a self antigen encoded in a DNAvector can anergize self-reactive T cells, and prevent an autoimmuneattack. Co-stimulation of T cells by DNA encoding self-antigens isimpaired, thus attenuating pathogenic T-cells. Our observations in theEAE suggest a model where DNA immunization can be utilized for treatmentof autoimmune disease.

EXAMPLE 2 Protection Against Autoimmune Disease with an Interleukin-4DNA Co-vaccine via Induction of T-helper 2 Cells and STAT6 Activation

[0099] The following example demonstrates that that co-vaccinating thegenes for the cytokine IL4 along with the gene for PLP₁₃₉₋₁₅₁ as twoseparate plasmids can provide protective immunity against EAE. Inaddition, a mechanism is proposed, in which functional IL4 expressedfrom the DNA vaccine acts locally on autoreactive T cells via activationof STAT6 to shift their cytokine profile to a Th2 type. These resultsshow the engineering of a novel method of treatment of autoimmunedisease that combines the antigen specific effects of DNA vaccinationalong with the beneficial effects of local gene delivery.

[0100] Results

[0101] The IL4 DNA vaccine produces IL4 protein. In order to constructthe IL4 DNA vaccine, the complete coding sequence for IL4 was amplifiedby PCR from mouse spleen cDNA. This gene was cloned into the mammalianexpression vector pTargeT under control of the CMV promoter, and theplasmid was purified as described in the methods. In order todemonstrate that the IL4 cDNA construct can indeed produce full-lengthIL4 protein, an in vitro translation system was used. When the IL4 cDNAplasmid was transcribed and translated in vitro with ³⁵S-methionine andresolved by SDS-PAGE (polyacrylamide gel electrophoresis) andautoradiography, a single product of the correct size for mouse IL4 wasseen. A control reaction with vector DNA without insert or plasmidencoding PLP₁₃₉₋₁₅₁ produced no detectable product. The predictedmolecular weight for PLP₁₃₉₋₁₅₁ is approximately 1.5 kD and, therefore,would be extremely difficult to visualize by electrophoresis.

[0102] IL4 DNA vaccination causes activation of STAT6. In order todemonstrate that a DNA vaccine can act as a gene delivery vehicle, wewanted to explore the question of whether functional IL4 cytokine wasactually expressed from the DNA vaccine administered to the animal. IL4is known to act through the IL4 receptor to specifically activate STAT6,a member of the signal transducers and activators of transcriptionfamily (Takeda et al. (1996) Nature 380:627-30; Quelle et al. (1995) MolCell Biol 15:3336-43.

[0103] Mice were vaccinated intramuscularly on a once weekly basis withplasmid DNA encoding the IL4 cDNA as described in the methods. Draininglymph nodes were dissected one week after the last DNA vaccine. Proteinlysates were isolated from the lymph node cells, and probed for thepresence of activated STAT6 by Western blotting using a polyclonalantibody specific for the phosphorylated form of STAT6. As controls,mice were also vaccinated with pTargeT vector alone or with no DNA.Activated or phosphorylated STAT6 was only seen in lymph nodes from IL4DNA vaccinated mice. The phosphorylated STAT6 identified runs atapproximately 60 kD.

[0104] Identical results were obtained in a separate experiment in whichmice received three daily, rather than weekly, doses of the DNA vaccine.Mice were vaccinated intramuscularly with plasmid DNA on a daily basisfor three days. One day after the last DNA vaccine, protein lysates fromdraining lymph nodes were obtained and analyzed as above in ananti-phosphorylated STAT6 Western. A 60 kD band was seen only in thelymph node cells from IL4 DNA vaccinated mice.

[0105] Co-vaccination with DNA encoding IL4 and the PLP₁₃₉₋₁₅₁ minigeneprotects against EAE induction. In order to explore the effect ofmodifying the protection afforded by DNA immunization with the geneencoding PLP₁₃₉₋₁₅₁, we co-vaccinated mice with the genes for IL4 andPLP₁₃₉₋₁₅₁ as two separate plasmids. The murine IL4 gene was cloned intothe mammalian expression vector pTargeT under control of the CMVpromoter as described earlier. The gene encoding PLP₁₃₉₋₁₅₁ was obtainedas described above.

[0106] SJL/J mice were injected with 100 μg of each plasmidintramuscularly twice, at one-week intervals. Control mice were injectedwith vector alone or with PBS. Ten days after the last injection, themice were challenged for induction of EAE with the encephalitogenicpeptide PLP₁₃₉₋₁₅₁ emulsified in complete Freund's adjuvant (CFA). Asshown in Table 3, there is a significant decrease in the mean diseasescores of mice co-vaccinated with both the IL4 and PLP₁₃₉-₁₅₁ plasmidscompared to controls (see table for p values). There is also a decreasein the incidence of disease and mean peak disease severity with theco-vaccine compared to controls. The onset of disease was notsignificantly delayed compared to the control groups. No significantprotection from disease was seen in mice vaccinated only with DNAencoding IL4. TABLE 2 EAE disease severity in DNA vaccinated miceMean^(a) Percent Peak Mean Mean Mean Inci- Disease Score Score Score DNAn dence Severity day 12 day 14 day 16 None 14 86 2.3 ± 0.3 1.6 ± 0.4 1.2± 0.2 0.7 ± 0.3 pTargeT 15 93 2.4 ± 0.2 1.6 ± 0.3 1.7 ± 0.2 1.1 ± 0.2IL4 15 80 2.7 ± 0.3 1.4 ± 0.3 1.1 ± 0.2 0.4 ± 0.2 IL4 & 15 53 1.6 ± 0.30.8 ± 0.3 0.7 ± 0.3 0.5 ± 0.2 PLP 139-151 (p < (p < (p < (p <0.0383)^(b) 0.0494) 0.0075) 0.0411)

[0107] Co-vaccination with DNA encoding IL4 rescues the T cellproliferative responses in PLP₁₃₉₋₁₅₁ DNA vaccinated animals. Mice thatwere vaccinated with DNA and challenged for disease induction withpeptide PLP₁₃₉₋₁₅₁ were sacrificed after recovery from the initial acutedisease. Draining lymph node cells (LNC) were obtained from these miceand re-stimulated in vitro with the PLP₁₃₉₋₁₅₁ peptide to determinetheir proliferative responses. Furthermore, antigen specific T celllines were maintained from these LNC in order to analyze their cytokinesecretion profiles.

[0108] LNC were tested for their proliferative responses to the peptidePLP₁₃₉₋₁₅₁. There was no significant change in the proliferative patternof LNC from IL4 and PLP₁₃₉₋₁₅₁ co-DNA vaccinated mice compared tocontrol mice vaccinated with vector only. In contrast, LNC from micevaccinated only with PLP₁₃₉₋₁₅₁ DNA have a reduced proliferativecapacity. We have previously shown that these T cells are anergic(Example 1). Therefore, the addition of IL4 as a DNA co-vaccine is ableto rescue the anergy imposed by the PLP₁₃₉₋₁₅₁, DNA vaccine. Thus, adifferent mechanism of protection may be afforded by co-vaccination withIL4 DNA compared with vaccination with PLP₁₃₉₋₁₅₁ DNA alone.

[0109] Co-vaccination with DNA encoding IL4 changes the phenotype of Tcells into a Th2 type. PLP₁₃₉₋₁₅₁ specific T cells lines were isolatedand maintained in culture from mice challenged for disease inductionwith the peptide PLP₁₃₉₋₁₅₁ and previously vaccinated with variouscombinations of DNA. These T cell lines were tested for cytokineproduction after in vitro stimulation with the peptide PLP₁₃₉₋₁₅₁. Tcells from mice co-vaccinated with IL4 and PLP₁₃₉₋₁₅₁ DNA producedsignificantly higher amounts of IL4 (mean of 716±237 pg/ml vs.0.208±0.36 pg/ml from pTargeT vaccinated mice, p<0.0064) and IL10 (meanof 1073±221 pg/ml vs. 464±44 pg/ml from pTargeT vaccinated mice,p<0.0151) compared to T cells from control mice. In addition, T cellsfrom the IL4 and PLP₁₃₉₋₁₅₁ DNA co-vaccinated mice produced loweramounts of IFNγ compared to control T cells (mean of 1389±108 pg/ml vs.6689±85 pg/mi from pTargeT vaccinated mice, p<0.0001). Thus, T cellsisolated from the co-vaccinated and protected mice produce more Th2 typecytokines compared to control T cells. As reported above, T cells frommice vaccinated with PLP₁₃₉₋₁₅₁ DNA alone had a reduced amount of IFNγ,but did not undergo a Th2 shift.

[0110] Protection from EAE in IL4 and PLP₁₃₉₋₁₅₁ co-DNA vaccinated micecan be transferred by T cells. The T cells derived from miceco-vaccinated with both IL4 DNA and PLP₁₃₉₋₁₅₁ DNA, which maintainedproliferative capacity but underwent a Th2 shift, were then tested forthe capacity to transfer protection. Mice were immunized with theencephalitogenic peptide PLP₁₃₉₋₁₅₁ emulsified in CFA, and eight dayslater 10 million T cells were injected intravenously into each mouse.Animals were then followed for disease phenotype. Control T cells thatare specific for PLP₁₃₉₋₁₅₁ and known to induce EAE were also injectedas a control. Mice injected with T cells derived from the co-vaccinatedmice had reduced incidence (1/5 mice compare to 4/5 mice in thecontrols) and reduced disease scores compared with control T cellinjected mice. These results indicate that the protective effectachieved by IL4 and PLP₁₃₉₋₁₅₁ DNA co-vaccination can be transferred tonaive animals by antigen specific Th2 cells.

[0111] Discussion

[0112] This example demonstrates a novel method of protective immunitywhich combines the effects of DNA vaccination and local gene delivery.First we demonstrated that the IL4 genetic vaccine delivers functionalIL4. After confirming that full-length IL4 is indeed expressed in vitrofrom the DNA construct used for the vaccination, we then showed thatSTAT6 is activated in draining lymph node cells by the IL4 DNA vaccine.Because STAT6 is specifically activated by IL4, we believe that the mostlikely conclusion is that IL4 is produced from the DNA vaccineadministered and that it interacts with IL4 receptor on lymph nodecells, which in turn causes the activation of STAT6 downstream of thereceptor. The phosphorylated STAT6 identified in the present study isapproximately 60 kD. Although the predominant isoform of STAT6 describedin the literature is 100 kD, other isoforms have been described in mouseimmune tissues (Quelle et al., 1995). Furthermore, a recent studydemonstrated the existence of a 65 kD isoform in mouse mast cells(Sherman et al., 1999). The IL4 delivered by the DNA genetic vaccineappears to specifically activate this isoform. We were not able todetect antibody responses against IL4 in the IL4 DNA vaccinated mice.Therefore, we postulate that the IL4 gene thus delivered and expressedis effective in generating protective immunity without induction of animmune response against IL4.

[0113] When mice were immunized with both the IL4 DNA vaccine and aseparate DNA vaccine for the self-peptide PLP₁₃₉₋₁₅₁, these mice wereprotected against induction of disease by the peptide PLP₁₃₉₋₁₅₁emulsified in CFA. The IL4 DNA vaccine alone did not provide significantprotection. When the cytokine profile of T cells from co-vaccinated andprotected mice were examined, a shift to a Th2 type of cytokinesecretion pattern was seen. Furthermore, these Th2 cells could transferprotection against disease induction in naive mice. We thus propose thatthe combination of the local delivery of IL4 and vaccination withPLP₁₃₉₋₁₅₁ DNA causes the antigen specific autoreactive T cells to shifttheir phenotype to a more protective Th2 type of response. These antigenspecific, protective T cells are then directed to sites of myelin damageand attenuate the pathogenic autoimmune response.

[0114] A possible mechanism as to how this phenotypic shift could occuris that the IL4 and the PLP₁₃₉₋₁₅₁ DNA vaccines are taken up by antigenpresenting cells (APC's) at the site of administration of the vaccines.The PLP₁₃₉₋₁₅₁ peptide is expressed in the APC's and presented on MHCclass II to antigen specific T cells that are thus recruited. The APC'salso express IL4, which is secreted locally during the APC and T cellinteraction. This secreted IL4 then causes the phenotype of the antigenspecific T cell to assume a more Th2 type of phenotype. This model iscompatible with earlier studies that showed that T cells grown inculture can be caused to assume a more Th2 type of phenotype by growthin the presence of IL4 (Macatonia et al. (1993) Int Immunol 5:1119-28).

[0115] Previous studies have demonstrated that professional APC's eitherpresent at the site of administration or recruited from the bone marrowcan take up the naked DNA and travel to lymphoid organs (Chattergoon etal. (1998) J Immunol 160:5707-18). It is possible that two separate oreven distant APC's take up the two different plasmids. We believe,however, that it is the local microenvironment during the APC and T cellinteraction that is important since no detectable increase in serum IL4was seen in the IL4 DNA vaccinated mice. As a method of delivery of apotentially adverse gene product, such as a cytokine at high doses, thistechnique could be desirable over traditional gene therapy methods sincethe gene delivered acts locally rather than systemically.

[0116] DNA vaccines have proven to be effective in protecting againstsome animal models of autoimmune disease. One of the many advantages ofDNA vaccines over traditional treatments of autoimmune disease is theability to easily modify the treatment vehicle. We have shown here thatwith the addition of a genetically delivered IL4 cytokine to thePLP₁₃₉₋₁₅₁ DNA vaccine, we can protect against EAE and, further, drivethe protective response to a more Th2 type. The addition of IL4 as a DNAco-vaccine rescues the anergy imposed by the PLP₁₃₉₋₁₅₁ DNA vaccine, anddrives the response to a Th2 phenotype. This mechanism of protectionafforded by co-vaccination with IL4 DNA compared with vaccination withPLP₁₃₉₋₁₅₁ DNA alone, may have particular advantages. This techniquecould prove beneficial in the treatment of other autoimmune diseases.Immunization against the antigens that trigger those autoimmune diseasescaused by Th1 autoreactive cells, diseases such as multiple sclerosis,juvenile diabetes and rheumatoid arthritis, would be conditions whereco-vaccination with DNA encoding IL-4 might prove beneficial. Inconclusion, the data presented here imply a powerful and novel tool,namely the combination of local gene delivery and antigen specific DNAvaccination, that could be applied universally to all DNA vaccines.

[0117] Experimental Procedures

[0118] Animals. Six- to eight-week-old female SJL/J mice were purchasedfrom The Jackson Laboratory (Bar Harbor, Me.).

[0119] Peptides. Peptides were synthesized on a peptide synthesizer(model 9050; MilliGen, Burlington, Mass.) by standard9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by HPLC.Structures were confirmed by amino acid analysis and mass spectroscopy.Peptides used in these experiments were: (SEQ ID NO:5) PLP₁₃₉₋₁₅₁(HSLGKWLGHPDKF) and (SEQ ID NO:15) HSVP16 P45(DMTPADALDDRDLEM).

[0120] DNA vaccines. A minigene encoding PLP₁₃₉₋₁₅₁ was constructed asdescribed above. The murine IL4 gene was cloned by PCR from spleen cDNA(Clontech, Palo Alto, Calif.) by use of the following PCR primers: (SEQID NO:16) 5′-CGCGGATCCTTGATGGGTCTCAACCCCCAGCTAGTTGTC-3′ and (SEQ ID NO:17) 5′-ACGCTCGAGGTACTACGAGTAATCCATTTGCATGATGC-3′. Both of theseconstructs were cloned into the multiple cloning region of the pTargeTvector (Promega, Madison, Wis.), driven by the CMV promoter. Correctclones were confirmed by automated DNA sequencing. Purification of theplasmid DNA was performed with the use of the Qiagen Endo-free Mega Prepkit (Qiagen, Santa Clarita, Calif.). Purity of the plasmid DNA wasconfirmed by UV spectrophotometry and agarose gel electrophoresis. OnlyDNA with a 260 nm/280 nm absorbance ratio of greater than 1.7 was used.

[0121] In vitro translation. DNA constructs used for DNA vaccinationwere tested for the production of the correctly sized product by an invitro translation assay. Approximately 1 μg of plasmid DNA was incubatedfor 2 hours at 30° C. in a 50 μl volume containing the following: 25 μlof TNT rabbit reticulocyte lysate (Promega Corp., Madison, Wis.), 2 μlof TNT reaction buffer (Promega Corp., Madison, Wis.), 1 μl TNT T7 RNApolymerase (Promega Corp., Madison, Wis.), 1 μl of a 1 mM amino acidmixture minus methionine (Promega Corp., Madison, Wis.), 4 μl of³⁵S-methionine at 10 mCi/ml (Amersham Life Sciences Inc., ArlingtonHeights, Ill.), and 1 μl of RNasin ribonuclease inhibitor at 40 U/μl(Promega Corp., Madison, Wis.). A 3 μl volume of the products of thisreaction was mixed with SDS-sample buffer and run on an 18% SDSpolyacrylamide gel. After drying, the gel was then exposed toautoradiography film.

[0122] STAT6 Westerns. After dissection of draining lymph nodes from DNAvaccinated mice, the tissues were mechanically homogenized in 1 ml ofthe following buffer: 0.1 M NaCl, 0.01 M Tris-HCL pH7.4, 0.001 M EDTA, 1μg/ml aprotinin, 1.6 μM Pefabloc SC (Boehringer Mannheim, Indianapolis,Ind.). 0.5 ml of the resultant lysate was used in a BCA protein assay(Pierce, Rockford, Ill.) in order to determine the total proteinconcentration. The remaining 0.5 ml was added to 0.25 ml of 3×SDSloading buffer (New England Biolabs, Beverly, Mass.) containing DTT at afinal concentration of 0.04 M. The products were resolved on a 4-15%gradient SDS-PAGE gel (Bio-Rad, Hercules, Calif.). Prestained markerswere used to determine the molecular weights (Bio Rad, Hercules,Calif.). After electrophoresis, the gels were blotted to PVDF membranes(Amersham Life Sciences Inc., Arlington Heights, Ill.) at constantvoltage of 100 V in 25 mM Tris, 192 mM glycine and 20% (v/v) methanol asthe transfer buffer. The membranes were blocked for 1 hour at roomtemperature with Tris buffered saline (TBS), 0.1% Tween 20, and 20%non-fat dry milk. After washing the membranes with TBS and 0.1% Tween20, the membranes were hybridized overnight at 4° C. with anti-phosphoSTAT6 antibody (New England Biolabs, Beverly, Mass.) diluted 1:1000 inTBS, 0.1% Tween 20, 5% BSA. The membranes were then processed as in theECL Plus protocol (Amersham Life Sciences Inc., Arlington Heights, Ill.)for visualization of the bands by chemiluminescence. The membranes werestripped by incubation in 100 mM β-mercaptoethanol, 2% (w/v) SDS, and62.5 mM Tris-HCL pH 7.4 for 30 minutes at 60° C. These same membraneswere then probed with an antibody against mouse CD3ζ (Pharmingen, SanDiego, Calif.) as a control to verify equal loading of the lanes.

[0123] DNA immunization protocol Animals were injected in the leftquadriceps with 0.1 ml of 0.25% bupivicaine-HCL (Sigma, St. Louis, Mo.)in PBS. Two and 9 days later, mice were injected with 100 μg of plasmidDNA (at a concentration of 1 mg/ml in PBS) in the same muscle. Animalsreceiving a co-vaccine received two separate injections of each plasmidDNA.

[0124] EAE induction. Seven to 10 days after the final DNA vaccine, EAEwas induced in mice with 100 μg of PLP₁₃₉₋₁₅₁ peptide. The peptide wasdissolved in PBS at a concentration of 2 mg/ml and emulsified with anequal volume of incomplete Freund's adjuvant supplemented with 4 mg/mlheat killed mycobacterium tuberculosis H37Ra (Difco Laboratories,Detroit, Mich.). Mice were injected subcutaneously with 0.1 ml of thepeptide emulsion. Experimental animals were scored as follows: 1, tailweakness or paralysis; 2, hind limb weakness; 3, hind limb paralysis; 4,forelimb weakness or paralysis; and 5, moribund or dead animals.

[0125] Lymph node cell proliferation assays. After the acute phase ofdisease, draining lymph nodes were dissected and lymph node cells (LNC)were cultured in vitro for specific proliferative response to thePLP₁₃₉₋₁₅₁ peptide. LNC's were prepared in 96-well microtiter plates ina volume of 0.2 ml/well at a concentration of 2.5×10⁶ cells/ml. Theculture medium consisted of enriched RPMI (RPMI 1640 supplemented withL-glutamine [2 mM], sodium pyruvate [1 mM], nonessential amino acids[0.1 mM], penicillin [100 U/ml], streptomycin [0.1 mg/ml], 2-ME [5×10⁻⁵M]) supplemented with 1% autologous fresh normal mouse serum. Cultureswere incubated at 37° C. and after 72 hours, cells were pulsed for 18hours with 1 μCi/well of [³H]thymidine. The cells were then harvestedand counted in a beta counter.

[0126] Cytokine profile determination. T cell lines were establishedfrom LNC's derived from DNA vaccinated mice. These T cells were thentested for the production of various cytokines. 50×10³ T cells/ml wereincubated with 2.5×10⁶ irradiated syngenic APC's/ml in enriched RPMI and10% FCS. After 6 days of culture the supernatants were collected andtested by sandwich ELISA using standard ELISA kits (Pharmingen, SanDiego, Calif.).

EXAMPLE 3 Immunization with DNA Encoding an Immunodominant Peptide ofInsulin Prevents Diabetes in NOD Mice

[0127] The NOD mouse is an animal model of IDDM in which severalautoantigens, including insulin, have been identified. In this study itis proven that vaccination of NOD mice with DNA encoding animmunodominant peptide of insulin protects the animals from developingdiabetes. These results confirm that DNA vaccination has a protectiveeffect on autoimmunity and opens doors for novel therapies.

[0128] Materials and Methods

[0129] Animals. Three- to four-week-old female NOD mice were purchasedfrom Taconic Farms (Germantown, N.Y.) and maintained in the Departmentof Comparative Medicine at Stanford University.

[0130] Mice were tested weekly for glucosuria by Chemstrip (BoehringerMannheim Co., Indianapolis, Ind.), and diabetes was confirmed by plasmaglucose measurement using the One Touch II meter (Johnson & Johnson,Milpitas, Calif.). Animals having repeated plasma glucose levels greaterthan 250 mg/dl were considered diabetic.

[0131] Insulin peptide expression vectors. Overlapping sense andantisense oligonucleotide sequences encoding the A(7-21) and B(9-23)peptides of insulin were synthesized by the PAN facility at StanfordUniversity Medical Center. The nucleotide sequence of the insulin A (+)strand is (SEQ ID NO:18) 5′ CCGGAATTCGCCATGTGCACGTCAATCTGTTCACTGTACCAGCTAGAGAACTACTGCAACTAGTCTAQGAGC-3′; the sequence of the insulin B (+)strand is (SEQ ID NO:19) 5′-CCGGAATTCGCCATGAGCCACCTAGTAGAAGCACTAACCTCGTATGCGGCGAACGAGGTTAGTCTAGAGC-3′. These were designed to incorporateEcoRI and Xbal restriction sites for cloning. The products were clonedinto the multiple cloning region of PcDNA3.1⁺ expression vector(Invitrogen, Carlsbad, Calif.). Purification of the plasmid DNA wascarried out using Qiagen Endo-free Mega-prep kits (Qiagen, Valencia,Calif.).

[0132] Protein and peptides. Whole porcine insulin was purchased fromSigma (St. Louis, Mo.). Insulin peptides were synthesized and HPLCpurified by the PAN facility at Stanford University. The amino acidsequence of the insulin A (7-21) peptide is (SEQ ID NO:20)CTSICSLYQLENYCN; the sequence of insulin B (9-23) is (SEQ ID NO:21)SHLVEALYLVCGERG. The control peptide “p43” is derived from Bacillussubtilis hyp protein X13 and has the sequence (SEQ ID NO:22)RKVVTDFFKNIPQRI.

[0133] DNA Immunization Protocol. Experimental animals were injected at3 to 4 weeks of age in the quadricep with 0.1 ml of 0.25%bupivicaine-HCL (Sigma, St. Louis, Mo.) in PBS (0.05 ml per quadricep).Two days following, mice were injected with 0.05 ml of plasmid DNA at1.0 mg/ml in each quadricep. The plasmid DNA was injected two more timesat ten-day intervals.

[0134] Histology. The pancreata were removed from experimental andcontrol animals, fixed in 10% formaldehyde, and embedded in paraffin.Thin sections at three levels, 50 μm apart, were cut for staining withhematoxylin and eosin. The severity of infiltration was assessed bylight microscopy. Three and five animals from each group were analyzedfor two individual experiments, respectively. At least 25 islets wereexamined per pancreas.

[0135] Proliferation Assays. Ten days after the third injection ofplasmid DNA, animals were sacrificed and their splenocytes tested invitro for proliferative responses to insulin peptides and to other isletantigen peptides. A non-relevant peptide p43 was used as a control.Cells were plated in flat-bottom 96-well microtiter plates in a volumeof 0.2 ml per well at a concentration of 2.5×10⁶ cells per ml. Tissueculture media for the assay consisted of RPMI 1640 supplemented withL-glutamine (2 mM), sodium pyruvate (1 mM) nonessential amino acids (0.1mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2-ME (5×10⁻⁵ M),and 1% autologous fresh normal mouse serum. After 72 hours of incubationat 37 ° C., cells were pulsed with 1 μCi/well of [³H]thymidine for anadditional 18 hours. Plates were harvested and [³H] thymidineincorporation was measured in a scintillation counter.

[0136] Serum Antibody ELISAs. Polystyrene 96-well microtiter plates werecoated with 100 μl peptide or protein at a concentration of 10 μg/ml inPBS. Plates were washed and blocked with PBS containing 5% FCS for 1hour at room temperature. Diluted serum samples from vaccinated ornon-treated animals was added and incubated overnight at 4° C. Afterwashing, goat anti-mouse IgG conjugated to alkaline phosphatase(Southern Biotechnology Associates, Birmingham, Ala.) was added, andplates incubated for 1 hour at 37° C. After addition of the enzymesubstrate, plates were read at 405 nm in an ELISA reader.

[0137] Real Time Quantitative PCR analysis of cytokine mRNA. Five daysafter the second injection, pancreatic lymph nodes were harvested andsingle cell suspension prepared. Ten million cells from each group wereplated in 1.5 mL final volume with 10 μg/mL insulin B peptide. After 72hours cells were collected and pelleted for RNA extraction using theRNeasy kit (Quiagen, Valencia, Calif.). The RNA was treated with DNaseto remove all genomic DNA and reverse transcribed with MultiScribereverse transcriptase (PE Applied Biosystems, Foster City, Calif.) inthe presence of hexamers, according to manufacturer's instructions.

[0138] Real time quantitative PCR was carried out for IL-4, IFN-γ,IL-10, TGF-β, and ribosomal RNA (internal control) in the ABI Prism 7700sequence detector, which contains a GeneAmp PCR system R600 (PE AppliedBiosystems). The probes were labeled with the fluorescent reporter dyeFAM (6-carboxyfluorescein, covalently linked to the 5′ end of theoligonucleotide) and a quencher, TAMRA. The primer and probe sequencesused were the following: (SEQ ID NO:23) 5′ IL-4 primer, CATCGGCATTTTGAA;(SEQ ID NO:24) 3′ IL-4 primer, CGTTTGGCACATCCATCTCC; IL-4 probe, (SEQ IDNO:25) CACAGGAGAAGGGACGCCATGCA; 5′ IFN-γ primer, (SEQ ID NO:26)TCCTGCGGCCTAGCTCTGA; 3′ IFN-γ primer, (SEQ ID NO:27) GCCATGAGGAAGAGCT;IFN-γ probe, (SEQ ID NO:28) ACAATGAACGCTACACACTGCATCTTGGC; 5′ IL-10primer, (SEQ ID NO:29) TGCAGCAGCTCAGAGGGTTC; 3′ IL-10 primer, (SEQ IDNO:30) CTGGCCACAGTTTTCAGGGA; IL-10 probe, (SEQ ID NO:31)CCTACTGTCATCCCCCAGCCGCTTC; 5′ TGF-β primer, (SEQ ID NO:32)GCAACATGTGGAACTCTACCAGAA; 3′ TGF-β primer, (SEQ ID NO:33)GACGTCAAAAGACAGCCACTC; TGF-β probe, (SEQ ID NO:34)ACCTTGGTAACCGGCTGCTGACCC. All reactions were performed using the TaqManGold PT-PCR kit according to the manufacturer's instructions (PE AppliedBiosystems). For the different runs cDNA corresponding to 5 ng of totalRNA was used. A normalization to ribosomal RNA was performed for eachsample.

[0139] Statistical analysis. Disease was compared using an Analysis ofMaximum Likelihood Estimate and incidence rate. Proliferationmeasurements were compared using an F-ratio and student's t-test.

[0140] Results

[0141] NOD mice immunized with plasmid encoding the insulin B chainpeptide 9-23 are protected from diabetes. To test the efficacy of aninsulin DNA vaccine in the NOD mouse model, groups of 10, 4-week-old NODmice were injected with the DNA vaccine constructs and monitored fordiabetes weekly, as determined by glucosuria and hyperglycemia, for >30weeks. Results represent two independent experiments. Animals weremonitored for glucosuria twice weekly. Diabetes was established by twoconsecutive readings >250 mg/dl, and confirmed by blood glucosemeasurement. Data shown represents 10+10 mice studied over twoexperiments.

[0142] In the untreated and plasmid control (PcDNA) injected groups, 70%of the mice developed diabetes by 34 weeks of age (FIG. 5). In theinsB-PcDNA injected group, however, only 20% developed diabetes by thesame age (p=0.02 by X² analysis). Furthermore, the onset of disease wasmarkedly delayed in this group as well, from <14 weeks for the firstanimal to become diabetic in the untreated group, to >17 weeks for theinsB-PcDNA vaccinated group. The diabetes incidence rate for the PcDNAand untreated control groups was 3 times the rate for the insB-PcDNAgroup (0.035 and 0.036 for the PcDNA and untreated groups, respectively,compared to 0.012 for insB-PcDNA group.)

[0143] In InsB-PcDNA vaccinated NOD mice, insulitis coexists withprotection. Pancreata were removed from immunized and control animals at7 weeks of age, a time at which the initial infiltration of some isletsis clearly visible by histological staining of NOD pancreata. A minimumof twenty-five islets each for five animals per group were scored forinsulitis. Staining of pancreata from older (16-week-old) mice yieldedsimilar results. Although animals injected with insulin DNA showed noclinical signs of diabetes, islet infiltration (insulitis) was visibleat levels comparable to that seen in the control animals. Hencevaccination with insulin DNA did not affect gross trafficking oflymphocytes to the islets of Langerhans.

[0144] Proliferative responses of InsB-PcDNA Vaccinated NOD splenocytesagainst insulin are unaltered compared to controls. Spleens wereharvested from immunized animals 10 days following the thirdimmunization and tested for proliferative responses against insulin. Wefound a modest but not significant increase in proliferation byInsB-PcDNA immunized splenocytes compared to controls, which may reflectpriming of the small population of insulin-specific cells. Nonetheless,these results indicate that the mechanism of protection from IDDM is notdependent on induction of anergy in insulin-specific cells.

[0145] Insulin-specific antibodies are not induced by InsB-PcDNAvaccination. We tested whether DNA immunization induced antibodiesagainst the insulin peptide, or against other NOD autoantigens. Micewere bled at early (8 weeks) and late (25 weeks) time points, and theserum tested by ELISA for antibodies against whole insulin, insulin B(9-23), insulin A (7-21), GAD65, and Hsp60. We found no differencesbetween groups in antibody levels against the insulin B peptide, noragainst any of the other candidate antigens.

[0146] Immunization with Insulin B (9-23) DNA induces anantigen-specific response in the pancreatic lymph nodes. In order todetect antigen-specific responses in vitro, we used quantitative PCR toassess levels of cytokine mRNA production (FIG. 6). Pancreatic lymphnode cells from NOD mice vaccinated twice at a ten day interval witheither PcDNA or InsB-PcDNA were harvested 5 days after the secondinjection. Cells were cultured in the presence of insulin B (9-23)peptide for 72 hours, then pelleted for quantitative PCR analysis ofcytokine mRNA levels.

[0147] In three independent experiments, groups of animals were injectedtwice with either the insB-PcDNA or the PcDNA control plasmid. Five daysafter the second injection, pancreatic lymph nodes were harvested andsingle-cell suspension plated with 10 μg/mL insulin B (9-23) peptide.After 72 hours the cells were pelleted, and subjected to quantitativePCR analysis for IL-4, TGF-β, IL-10, and IFN-γ message levels.Quantitative PCR comparison of cytokine message levels in pancreaticlymph node cells showed a significant reduction in IFN-γ and IL-10levels in the insB-PcDNA vaccinated animals compared to PcDNA-vaccinatedcontrols. IFN-γ levels from insB-PcDNA-vaccinated lymph nodes were 38%that of PcDNA vaccinated lymph nodes (p<0.05) in response to insulin Bpeptide stimulation. Furthermore, IL-10 levels in InsB-PcDNA vaccinatedmice were 30% of PcDNA control levels (p<0.01). Changes in mRNA levelsof IL-4 and TGF-β were not significant over the three experiments.

[0148] The above data demonstrate the successful vaccination of NOD micewith insulin B (9-23) DNA to confer protection from diabetes. The effectis specific to DNA encoding immunogenic insulin, since empty plasmidalone, or DNA encoding a non-immunogenic peptide of insulin, did nothave a significant effect on disease. Bacterial CpG motifs could notaccount for the protection, since the plasmid encoding insulin A (7-21),which was identical in length and contained the same number of CpGs, didnot alter disease incidence significantly. Disease onset wassubstantially delayed in the InsB-PcDNA vaccinated mice that did becomediabetic, reiterating the protective potential of DNA vaccination.Protection from diabetes appeared to be associated with down regulationof IFN-γ and IL-10 in pancreatic lymph node cells in response to theinsulin B peptide encoded in the vaccine.

[0149] Insulitis was not abolished in protected animals, indicating thatDNA vaccination did not reduce the gross trafficking of cells to theislets, although there may be a selective alteration in lymphocytetrafficking. Furthermore, the infiltrate was relatively non-destructivewithin the time of analysis (up to 16 weeks of age), as most InsB-PcDNAvaccinated mice did not become diabetic. This outcome is consistent withdata describing polypeptide based immunization with whole insulin orwith the B chain peptide. Regulation of diabetes does not necessarilytake place at the level of infiltration of the islets by lymphocytes,but rather, at the level of the actual destruction of theinsulin-secreting β cells.

[0150] There was no significant increase in T cell proliferativeresponses to insulin B peptide and whole insulin by splenocytes ofinsB-PcDNA vaccinated animals. This result indicates that insulin B(9-23) DNA vaccination in the NOD mice does not anergize or eliminate Tcells specific for the encoded peptide. Rather, it was found that cellsfrom insB-PcDNA immunized animals expressed substantially lower amountsof IFN-γ than did cells from control vaccinated animals. This downregulation of IFN-γ secretion in response to activation correlatesstrongly with protection from diabetes, since IFN-γ is known to be acritical mediator of inflammation of the islets and of β celldestruction. The decrease in levels of IL-10 expression may alsocontribute to protection from disease.

[0151] These results suggest that DNA vaccination may be an effectivemethod of altering harmful immune responses in autoimmunity to conferprotection. Furthermore, DNA vaccination will be a powerful tool inmodulating disease.

1 34 1 2777 DNA Homo sapiens CDS (119)...(952) 1 gaattcggga aaagaccgaagaaggaggct ggagagacca ggatccttcc agctgaacaa 60 agtcagccac aaagcagactagccagccgg ctacaattgg agtcagagtc ccaaagac 118 atg ggc ttg tta gag tgctgt gca aga tgt ctg gta ggg gcc ccc ttt 166 Met Gly Leu Leu Glu Cys CysAla Arg Cys Leu Val Gly Ala Pro Phe 1 5 10 15 gct tcc ctg gtg gcc actgga ttg tgt ttc ttt ggg gtg gca ctg ttc 214 Ala Ser Leu Val Ala Thr GlyLeu Cys Phe Phe Gly Val Ala Leu Phe 20 25 30 tgt ggc tgt gga cat gaa gccctc act ggc aca gaa aag cta att gag 262 Cys Gly Cys Gly His Glu Ala LeuThr Gly Thr Glu Lys Leu Ile Glu 35 40 45 acc tat ttc tcc aaa aac tac caagac tat gag tat ctc atc aat gtg 310 Thr Tyr Phe Ser Lys Asn Tyr Gln AspTyr Glu Tyr Leu Ile Asn Val 50 55 60 atc cat gcc ttc cag tat gtc atc tatgga act gcc tct ttc ttc ttc 358 Ile His Ala Phe Gln Tyr Val Ile Tyr GlyThr Ala Ser Phe Phe Phe 65 70 75 80 ctt tat ggg gcc ctc ctg ctg gct gagggc ttc tac acc acc ggc gca 406 Leu Tyr Gly Ala Leu Leu Leu Ala Glu GlyPhe Tyr Thr Thr Gly Ala 85 90 95 gtc agg cag atc ttt ggc gac tac aag accacc atc tgc ggc aag ggc 454 Val Arg Gln Ile Phe Gly Asp Tyr Lys Thr ThrIle Cys Gly Lys Gly 100 105 110 ctg agc gca acg gta aca ggg ggc cag aagggg agg ggt tcc aga ggc 502 Leu Ser Ala Thr Val Thr Gly Gly Gln Lys GlyArg Gly Ser Arg Gly 115 120 125 caa cat caa gct cat tct ttg gag cgg gtgtgt act tgt ttg gga aaa 550 Gln His Gln Ala His Ser Leu Glu Arg Val CysThr Cys Leu Gly Lys 130 135 140 tgg cta gga cat ccc gac aag ttt gtg ggcatc acc tat gcc ctg acc 598 Trp Leu Gly His Pro Asp Lys Phe Val Gly IleThr Tyr Ala Leu Thr 145 150 155 160 gtt gtg tgg ctc ctg gtg ttt gcc tgctct gct gtg cct gtg tac att 646 Val Val Trp Leu Leu Val Phe Ala Cys SerAla Val Pro Val Tyr Ile 165 170 175 tac ttc aac acc tgg acc acc tgc cagtct att gcc ttc ccc agc aag 694 Tyr Phe Asn Thr Trp Thr Thr Cys Gln SerIle Ala Phe Pro Ser Lys 180 185 190 acc tct gcc agt ata ggc agt ctc tgtgct gac gcc aga atg tat ggt 742 Thr Ser Ala Ser Ile Gly Ser Leu Cys AlaAsp Ala Arg Met Tyr Gly 195 200 205 gtt ctc cca tgg aat gct ttc cct ggcaag gtt tgt ggc tcc aac ctt 790 Val Leu Pro Trp Asn Ala Phe Pro Gly LysVal Cys Gly Ser Asn Leu 210 215 220 ctg tcc atc tgc aaa aca gct gag ttccaa atg acc ttc cac ctg ttt 838 Leu Ser Ile Cys Lys Thr Ala Glu Phe GlnMet Thr Phe His Leu Phe 225 230 235 240 att gct gca ttt gtg ggg gct gcagct aca ctg gtt tcc ctg ctc acc 886 Ile Ala Ala Phe Val Gly Ala Ala AlaThr Leu Val Ser Leu Leu Thr 245 250 255 ttc atg att gct gcc act tac aacttt gcc gtc ctt aaa ctc atg ggc 934 Phe Met Ile Ala Ala Thr Tyr Asn PheAla Val Leu Lys Leu Met Gly 260 265 270 cga ggc acc aag ttc tgatcccccgtag aaatccccct ttctctaata 982 Arg Gly Thr Lys Phe * 275gcgaggctcc tctaaccaca cagcctacaa tgctgcgtct cccatcttaa ctctttgcct 1042ttgccaccaa ctggccctct tcttacttga tgagtgtaac aagaaaggag agtcttgcag 1102tgattaaggt ctctctttgg actctcccct cttatgtacc tcttttagtc attttgcttc 1162atagctggtt cctgctagaa atgggaaatg cctaagaaga tgacttccca actcgaagtc 1222acaaaggaat ggaggctcta attgaatttt caagcatctc ctgaggatca gaaagtaatt 1282tcttctcaaa gggtacttcc actgatggaa acaaagtgga aggaaagaag gtcaggtaca 1342gagaaggaat gtctttggtc ctcttgccat ctataggggc caaatatatt ctctttggtg 1402tacaaaatgg aattcattct ggtctctcta ttaccactga agatagaaga aaaaagaatg 1462tcagaaaaac aataagagcg tttgcccaaa tctgcctatt gcagctggga gaagggggtc 1522aaagcaagga tctttcaccc acagaaagag agcactgacc ccgatggcga tggactactg 1582aagccctaac tcagccaacc ttacttacag cataagggag cgtagaatct gtgtagacga 1642agggggcatc tggccttaca cctcgttagg gaagagaaac agggtgttgt cagcatcttc 1702tcactccctt ctccttgata acagctacca tgacaaccct gtggtttcca aggagctgag 1762aatagaagga aactagctta catgagaaca gactggcctg aggagcagca gttcctggtg 1822gctaatggtg taacctgaga tggccctctg gtagacacag gatagataac tctttggata 1882gcatgtcttt ttttctgtta attagttgtg tactctggcc tctgtcatat cttcacaatg 1942gtgctcattt catggggtat tatccattca gtcatcgtag gtgatttgaa ggtcttgatt 2002tgttttagaa tgatgcacat ttcatgtatt ccagtttgtt tattacttat ttggggttgc 2062atcagaaatg tctggagaat aattctttga ttatgactgt tttttaaact aggaaaattg 2122gacattaagc atcacaaatg atattaaaaa ttggctagtt gaatctattg ggattttcta 2182caagtattct gcctttgcag aaacagattt ggtgaatttg aatctcaatt tgagtaatct 2242gatcgttctt tctagctaat ggaaaatgat tttacttagc aatgttatct tggtgtgtta 2302agagttaggt ttaacataaa ggttattttc tcctgatata gatcacataa cagaatgcac 2362cagtcatcag ctattcagtt ggtaagcttc caggaaaaag gacaggcaga aagagtttga 2422gacctgaata gctcccagat ttcagtcttt tcctgttttt gttaactttg ggttaaaaaa 2482aaaaaaagtc tgattggttt taattgaagg aaagatttgt actacagttc ttttgttgta 2542aagagttgtg ttgttctttt cccccaaagt ggtttcagca atatttaagg agatgtaaga 2602gctttacaaa aagacacttg atacttgttt tcaaaccagt atacaagata agcttccagg 2662ctgcatagaa ggaggagagg gaaaatgttt tgtaagaaac caatcaagat aaaggacagt 2722gaagtaatcc gtaccttgtg ttttgttttg atttaataac ataacaaata accaa 2777 2 277PRT Homo sapiens 2 Met Gly Leu Leu Glu Cys Cys Ala Arg Cys Leu Val GlyAla Pro Phe 1 5 10 15 Ala Ser Leu Val Ala Thr Gly Leu Cys Phe Phe GlyVal Ala Leu Phe 20 25 30 Cys Gly Cys Gly His Glu Ala Leu Thr Gly Thr GluLys Leu Ile Glu 35 40 45 Thr Tyr Phe Ser Lys Asn Tyr Gln Asp Tyr Glu TyrLeu Ile Asn Val 50 55 60 Ile His Ala Phe Gln Tyr Val Ile Tyr Gly Thr AlaSer Phe Phe Phe 65 70 75 80 Leu Tyr Gly Ala Leu Leu Leu Ala Glu Gly PheTyr Thr Thr Gly Ala 85 90 95 Val Arg Gln Ile Phe Gly Asp Tyr Lys Thr ThrIle Cys Gly Lys Gly 100 105 110 Leu Ser Ala Thr Val Thr Gly Gly Gln LysGly Arg Gly Ser Arg Gly 115 120 125 Gln His Gln Ala His Ser Leu Glu ArgVal Cys Thr Cys Leu Gly Lys 130 135 140 Trp Leu Gly His Pro Asp Lys PheVal Gly Ile Thr Tyr Ala Leu Thr 145 150 155 160 Val Val Trp Leu Leu ValPhe Ala Cys Ser Ala Val Pro Val Tyr Ile 165 170 175 Tyr Phe Asn Thr TrpThr Thr Cys Gln Ser Ile Ala Phe Pro Ser Lys 180 185 190 Thr Ser Ala SerIle Gly Ser Leu Cys Ala Asp Ala Arg Met Tyr Gly 195 200 205 Val Leu ProTrp Asn Ala Phe Pro Gly Lys Val Cys Gly Ser Asn Leu 210 215 220 Leu SerIle Cys Lys Thr Ala Glu Phe Gln Met Thr Phe His Leu Phe 225 230 235 240Ile Ala Ala Phe Val Gly Ala Ala Ala Thr Leu Val Ser Leu Leu Thr 245 250255 Phe Met Ile Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys Leu Met Gly 260265 270 Arg Gly Thr Lys Phe 275 3 2139 DNA Homo sapiens CDS (37)...(552)3 gaaaacagtg cagccacctc cgagagcctg gatgtg atg gcg tca cag aag aga 54 MetAla Ser Gln Lys Arg 1 5 ccc tcc cag agg cac gga tcc aag tac ctg gcc acagca agt acc atg 102 Pro Ser Gln Arg His Gly Ser Lys Tyr Leu Ala Thr AlaSer Thr Met 10 15 20 gac cat gcc agg cat ggc ttc ctc cca agg cac aga gacacg ggc atc 150 Asp His Ala Arg His Gly Phe Leu Pro Arg His Arg Asp ThrGly Ile 25 30 35 ctt gac tcc atc ggg cgc ttc ttt ggc ggt gac agg ggt gcgcca aag 198 Leu Asp Ser Ile Gly Arg Phe Phe Gly Gly Asp Arg Gly Ala ProLys 40 45 50 cgg ggc tct ggc aag gac tca cac cac ccg gca aga act gct cactat 246 Arg Gly Ser Gly Lys Asp Ser His His Pro Ala Arg Thr Ala His Tyr55 60 65 70 ggc tcc ctg ccc cag aag tca cac ggc cgg acc caa gat gaa aacccc 294 Gly Ser Leu Pro Gln Lys Ser His Gly Arg Thr Gln Asp Glu Asn Pro75 80 85 gta gtc cac ttc ttc aag aac att gtg acg cct cgc aca cca ccc ccg342 Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro 9095 100 tcg cag gga aag ggg aga gga ctg tcc ctg agc aga ttt agc tgg ggg390 Ser Gln Gly Lys Gly Arg Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly 105110 115 gcc gaa ggc cag aga cca gga ttt ggc tac gga ggc aga gcg tcc gac438 Ala Glu Gly Gln Arg Pro Gly Phe Gly Tyr Gly Gly Arg Ala Ser Asp 120125 130 tat aaa tcg gct cac aag gga ttc aag gga gtc gat gcc cag ggc acg486 Tyr Lys Ser Ala His Lys Gly Phe Lys Gly Val Asp Ala Gln Gly Thr 135140 145 150 ctt tcc aaa att ttt aag ctg gga gga aga gat agt cgc tct ggatca 534 Leu Ser Lys Ile Phe Lys Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser155 160 165 ccc atg gct aga cgc tga aaacccacct ggttccggaa tcctgtcctc 582Pro Met Ala Arg Arg * 170 agcttcttaa tataactgcc ttaaaacttt aatcccacttgcccctgtta cctaattaga 642 gcagatgacc cctcccctaa tgcctgcgga gttgtgcacgtagtagggtc aggccacggc 702 agcctaccgg caatttccgg ccaacagtta aatgagaacatgaaaacaga aaacggttaa 762 aactgtccct ttctgtgtga agatcacgtt ccttcccccgcaatgtgccc ccagacgcac 822 gtgggtcttc agggggccag gtgcacagac gtccctccacgttcacccct ccacccttgg 882 actttctttt cgccgtggct cggcaccctt gcgcttttgctggtcactgc catggaggca 942 cacagctgca gagacagaga ggacgtgggc ggcagagaggactgttgaca tccaagcttc 1002 ctttgttttt ttttcctgtc cttctctcac ctcctaaagtagacttcatt tttcctaaca 1062 ggattagaca gtcaaggagt ggcttactac atgtgggagctttttggtat gtgacatgcg 1122 ggctgggcag ctgttagagt ccaacgtggg gcagcacagagagggggcca cctccccagg 1182 ccgtggctgc ccacacaccc caattagctg aattcgcgtgtggcagaggg aggaaaagga 1242 ggcaaacgtg ggctgggcaa tggcctcaca taggaaacagggtcttcctg gagatttggt 1302 gatggagatg tcaagcaggt ggcctctgga cgtcaccgttgccctgcatg gtggccccag 1362 agcagcctct atgaacaacc tcgtttccaa accacagcccacagccggag agtccaggaa 1422 gacttgcgca ctcagagcag aagggtagga gtcctctagacagcctcgca gccgcgccag 1482 tcgcccatag acactggctg tgaccgggcg tgctggcagcggcagtgcac agtggccagc 1542 actaaccctc cctgagaaga taaccggctc attcacttcctcccagaaga cgcgtggtag 1602 cgagtaggca caggcgtgca cctgctcccg aattactcaccgagacacac gggctgagca 1662 gacggcccct gtgatggaga caaagagctc ttctgaccatatccttctta acacccgctg 1722 gcatctcctt tcgcgcctcc ctccctaacc tactgacccaccttttgatt ttagcgcacc 1782 tgtgattgat aggccttcca aagagtccca cgctggcatcaccctccccg aggacggaga 1842 tgaggagtag tcagcgtgat gccaaaacgc gtcttcttaatccaattcta attctgaatg 1902 tttcgtgtgg gcttaatacc atgtctatta atatatagcctcgatgatga gagagttaca 1962 aagaacaaaa ctccagacac aaacctccaa atttttcagcagaagcactc tgcgtcgctg 2022 agctgaggtc ggctctgcga tccatacgtg gccgcacccacacagcacgt gctgtgacga 2082 tggctgaacg gaaagtgtac actgttcctg aatattgaaataaaacaata aactttt 2139 4 171 PRT Homo sapiens 4 Met Ala Ser Gln Lys ArgPro Ser Gln Arg His Gly Ser Lys Tyr Leu 1 5 10 15 Ala Thr Ala Ser ThrMet Asp His Ala Arg His Gly Phe Leu Pro Arg 20 25 30 His Arg Asp Thr GlyIle Leu Asp Ser Ile Gly Arg Phe Phe Gly Gly 35 40 45 Asp Arg Gly Ala ProLys Arg Gly Ser Gly Lys Asp Ser His His Pro 50 55 60 Ala Arg Thr Ala HisTyr Gly Ser Leu Pro Gln Lys Ser His Gly Arg 65 70 75 80 Thr Gln Asp GluAsn Pro Val Val His Phe Phe Lys Asn Ile Val Thr 85 90 95 Pro Arg Thr ProPro Pro Ser Gln Gly Lys Gly Arg Gly Leu Ser Leu 100 105 110 Ser Arg PheSer Trp Gly Ala Glu Gly Gln Arg Pro Gly Phe Gly Tyr 115 120 125 Gly GlyArg Ala Ser Asp Tyr Lys Ser Ala His Lys Gly Phe Lys Gly 130 135 140 ValAsp Ala Gln Gly Thr Leu Ser Lys Ile Phe Lys Leu Gly Gly Arg 145 150 155160 Asp Ser Arg Ser Gly Ser Pro Met Ala Arg Arg 165 170 5 13 PRTArtificial Sequence PLP139-151 5 His Ser Leu Gly Lys Trp Leu Gly His ProAsp Lys Phe 1 5 10 6 13 PRT Artificial Sequence PLP139-151 L144/R147 6His Ser Leu Gly Lys Leu Leu Gly Arg Pro Asp Lys Phe 1 5 10 7 14 PRTArtificial Sequence PLP178-191 7 Asn Thr Trp Thr Thr Cys Gln Ser Ile AlaPhe Pro Ser Lys 1 5 10 8 65 DNA Artificial Sequence PLP (178-191) 8ctggagacca gaatacctgg accacctgcc agtctattgc cttccctagc aagtctagat 60agcta 65 9 63 DNA Artificial Sequence PLP (139-151) 9 ctcgagaccatgcattgttt gggaaaatgg ctaggacatc ccgacaagtt ttctagatag 60 cta 63 10 63DNA Artificial Sequence PLP (139-151) L144/R147 10 ctcgagacca tgcattgtttgggaaaacta ctaggacgcc ccgacaagtt ttctagatag 60 cta 63 11 13 PRT Homosapiens 11 Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro 1 5 10 127 PRT Homo sapiens 12 Phe Lys Asn Ile Val Thr Pro 1 5 13 153 PRTArtificial Sequence amino acid sequence of a Th2 cytokine 13 Met Gly LeuThr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu Ala 1 5 10 15 Cys AlaGly Asn Phe Val His Gly His Lys Cys Asp Ile Thr Leu Gln 20 25 30 Glu IleIle Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys Thr Leu Cys 35 40 45 Thr GluLeu Thr Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr 50 55 60 Glu LysGlu Thr Phe Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr 65 70 75 80 SerHis His Glu Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala Gln Gln 85 90 95 PheHis Arg His Lys Gln Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg 100 105 110Asn Leu Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro Val Lys Glu Ala 115 120125 Asn Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met 130135 140 Arg Glu Lys Tyr Ser Lys Cys Ser Ser 145 150 14 614 DNAArtificial Sequence sequence encoding a Th2 cytokine 14 gatcgttagcttctcctgat aaactaattg cctcacattg tcactgcaaa tcgacaccta 60 tta atg ggtctc acc tcc caa ctg ctt ccc cct ctg ttc ttc ctg cta 108 Met Gly Leu ThrSer Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu 1 5 10 15 gca tgt gcc ggcaac ttt gtc cac gga cac aag tgc gat atc acc tta 156 Ala Cys Ala Gly AsnPhe Val His Gly His Lys Cys Asp Ile Thr Leu 20 25 30 cag gag atc atc aaaact ttg aac agc ctc aca gag cag aag act ctg 204 Gln Glu Ile Ile Lys ThrLeu Asn Ser Leu Thr Glu Gln Lys Thr Leu 35 40 45 tgc acc gag ttg acc gtaaca gac atc ttt gct gcc tcc aag aac aca 252 Cys Thr Glu Leu Thr Val ThrAsp Ile Phe Ala Ala Ser Lys Asn Thr 50 55 60 act gag aag gaa acc ttc tgcagg gct gcg act gtg ctc cgg cag ttc 300 Thr Glu Lys Glu Thr Phe Cys ArgAla Ala Thr Val Leu Arg Gln Phe 65 70 75 tac agc cac cat gag aag gac actcgc tgc ctg ggt gcg act gca cag 348 Tyr Ser His His Glu Lys Asp Thr ArgCys Leu Gly Ala Thr Ala Gln 80 85 90 95 cag ttc cac agg cac aag cag ctgatc cga ttc ctg aaa cgg ctc gac 396 Gln Phe His Arg His Lys Gln Leu IleArg Phe Leu Lys Arg Leu Asp 100 105 110 agg aac ctc tgg ggc ctg gcg ggcttg aat tcc tgt cct gtg aag gaa 444 Arg Asn Leu Trp Gly Leu Ala Gly LeuAsn Ser Cys Pro Val Lys Glu 115 120 125 gcc aac cag agt acg ttg gaa aacttc ttg gaa agg cta aag acg atc 492 Ala Asn Gln Ser Thr Leu Glu Asn PheLeu Glu Arg Leu Lys Thr Ile 130 135 140 atg aga gag aaa tat tca aag tgttcg agc tga atattttaat ttatgagttt 545 Met Arg Glu Lys Tyr Ser Lys CysSer Ser * 145 150 ttgatagctt tattttttaa gtatttatat atttataact catcataaaataaagtatat 605 atagaatct 614 15 15 PRT Artificial Sequence HSVP16 P45 15Asp Met Thr Pro Ala Asp Ala Leu Asp Asp Arg Asp Leu Glu Met 1 5 10 15 1639 DNA Artificial Sequence oligonucleotide primer 16 cgcggatccttgatgggtct caacccccag ctagttgtc 39 17 38 DNA Artificial Sequenceoligonucleotide primer 17 acgctcgagg tactacgagt aatccatttg catgatgc 3818 72 DNA Artificial Sequence insulin A (+) strand 18 ccggaattcgccatgtgcac gtcaatctgt tcactgtacc agctagagaa ctactgcaac 60 tagtctanga gc72 19 70 DNA Artificial Sequence insulin B (+) strand 19 ccggaattcgccatgagcca cctagtagaa gcactaacct cgtatgcggc gaacgaggtt 60 agtctagagc 7020 15 PRT Artificial Sequence insulin A (7-21) peptide 20 Cys Thr SerIle Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 1 5 10 15 21 15 PRTArtificial Sequence insulin B (9-23) peptide 21 Ser His Leu Val Glu AlaLeu Tyr Leu Val Cys Gly Glu Arg Gly 1 5 10 15 22 15 PRT ArtificialSequence control peptide p43 derived from Bacillus subtilis hype proteinX13 22 Arg Lys Val Val Thr Asp Phe Phe Lys Asn Ile Pro Gln Arg Ile 1 510 15 23 15 DNA Artificial Sequence IL-4 primer 23 catcggcatt ttgaa 1524 20 DNA Artificial Sequence IL-4 primer 24 cgtttggcac atccatctcc 20 2523 DNA Artificial Sequence IL-4 probe 25 cacaggagaa gggacgccat gca 23 2619 DNA Artificial Sequence IFN-gamma primer 26 tcctgcggcc tagctctga 1927 16 DNA Artificial Sequence IFN-gamma primer 27 gccatgagga agagct 1628 29 DNA Artificial Sequence IFN-gamma probe 28 acaatgaacg ctacacactgcatcttggc 29 29 20 DNA Artificial Sequence IL-10 primer 29 tgcagcagctcagagggttc 20 30 20 DNA Artificial Sequence IL-10 primer 30 ctggccacagttttcaggga 20 31 25 DNA Artificial Sequence IL-10 probe 31 cctactgtcatcccccagcc gcttc 25 32 24 DNA Artificial Sequence TGF-beta primer 32gcaacatgtg gaactctacc agaa 24 33 21 DNA Artificial Sequence TGF-betaprimer 33 gacgtcaaaa gacagccact c 21 34 24 DNA Artificial SequenceTGF-beta probe 34 accttggtaa ccggctgctg accc 24

What is claimed is:
 1. A method for treating an autoimmune disease in amammalian host, the method comprising: introducing into said mammalianhost a DNA expression cassette comprising: a sequence encoding at leasta portion of an autoantigen associated with a pro-inflammatory, Th1-typeT cell response, under the regulatory control of a promoter that isactive in said mammalian host, under conditions wherein said expressioncassette is incorporated into cells of said host and said sequence isexpressed; wherein the pro-inflammatory response of T cells that respondto said autoantigen is decreased.
 2. The method of claim 1, furthercomprising introducing into said mammalian host a DNA expressioncassette comprising a sequence encoding a Th2 cytokine under theregulatory control of a promoter that is active in said mammalian host,under conditions wherein said expression cassette is incorporated intocells of said host and said sequence is expressed.
 3. The method ofclaim 2, wherein said Th2 cytokine is IL4.
 4. The method of claim 3,wherein said IL4 is human IL4.
 5. The method of claim 2, wherein saidsequence encoding said Th2 cytokine and said sequence encoding at leasta portion of an autoantigen are present on a single DNA construct. 6.The method of claim 2, wherein said sequence encoding said Th2 cytokineand said sequence encoding at least a portion of an autoantigen arepresent on separate DNA constructs.
 7. The method of claim 2, whereinsaid expression construct encoding said Th2 cytokine and said expressionconstruct encoding at least a portion of an autoantigen areco-formulated.
 8. The method of claim 2, wherein said expressionconstruct encoding said Th2 cytokine and said expression constructencoding at least a portion of an autoantigen are independentlyformulated.
 9. The method of claim 1, wherein said autoantigen is amyelin protein.
 10. The method of claim 9, wherein said myelin proteinis proteolipid protein.
 11. The method of claim 9, wherein said myelinprotein is myelin basic protein.
 12. The method of claim 9, wherein saidmyelin protein is myelin oligodendrocyte protein.
 13. The method ofclaim 9, wherein said myelin protein is myelin associated protein. 14.The method of claim 9, wherein said autoimmune disease is ademyelinating disease.
 15. The method of claim 14, wherein saiddemyelinating disease is experimental autoimmune encephalitis.
 16. Themethod of claim 14, wherein said demyelinating disease is multiplesclerosis.
 17. The method of claim 1, wherein a plasmid comprises saidDNA expression cassette.
 18. The method of claim 1, wherein saidpro-inflammatory response includes expression of IL-2, γ-interferon orIL-15.
 19. A vaccine comprising: a DNA construct comprising a sequenceencoding at least a portion of an autoantigen associated with apro-inflammatory, Th1-type T cell response, under the regulatory controlof a promoter that is active in a mammalian host; and a sequenceencoding a Th2 cytokine under the regulatory control of a promoter thatis active in a mammalian host.
 20. The vaccine of claim 19, wherein saidTh2 cytokine is IL4.
 21. The vaccine of claim 20, wherein said IL4 ishuman IL4.
 22. The vaccine of claim 19, wherein said sequence encodingsaid Th2 cytokine and said sequence encoding at least a portion of anautoantigen are present on a single DNA construct.
 23. The vaccine ofclaim 19, wherein said sequence encoding said Th2 cytokine and saidsequence encoding at least a portion of an autoantigen are present onseparate DNA constructs.
 24. The vaccine of claim 19, wherein saidautoantigen is a myelin protein.
 25. The vaccine of claim 19, whereinsaid myelin protein is proteolipid protein.
 26. The vaccine of claim 19,wherein said myelin protein is myelin basic protein.
 27. The vaccine ofclaim 19, wherein said myelin protein is myelin oligodendrocyte protein.28. The vaccine of claim 19, wherein said myelin protein is myelinassociated protein.